JP2023554389A - Method for producing recombinant adeno-associated virus virus particles - Google Patents

Abstract

Provided herein are improved methods for producing recombinant virus particles. In some embodiments, the methods for producing recombinant virus particles described herein include providing a suspension cell culture comprising a population of cells capable of producing virus particles; transferring a first volume of the polynucleotide:transfection reagent complex to a suspension culture to transfect the polynucleotide:transfection reagent complex; and transferring a second volume of the polynucleotide:transfection reagent complex to the suspension culture. and transferring the first and second volumes of polynucleotide:transfection reagent complexes is performed simultaneously or sequentially in any order. In some embodiments, the recombinant viral particles are recombinant AAV (rAAV) particles. [Selection diagram] None

Description

The present disclosure relates to a method of producing recombinant virus particles on a large scale in suspension cell culture, the method comprising transfecting cells in the culture.

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit from U.S. Patent Application No. 63/126,405, filed December 16, 2020, which is incorporated herein by reference in its entirety. Ru.

Background Recombinant adeno-associated virus (AAV)-based vectors are currently the most widely used gene therapy products in development. The use of the rAAV vector system is preferred, in part, because of the lack of disease associated with wild-type virus, the ability of AAV to transduce both non-dividing and dividing cells, and the fact that it has been shown in clinical trials. Consequently, long-term and robust transgene expression has been observed, indicating great potential for delivery in gene therapy indications. In addition, the various natural and recombinant rAAV vector serotypes help specifically target different tissues, organs, and cells and evade any pre-existing immunity to the vector, thus making it an attractive candidate for AAV-based gene therapy. Expand therapeutic applications. Before replication-defective virus (e.g., AAV)-based gene therapy can be more widely adopted into late clinical stages and commercial use, there is a need to develop new methods for large-scale production of recombinant virus particles. There is.

Therefore, there is a need in the art to improve the productivity and yield of methods for producing rAAV particles on a large scale.

Brief Overview In one aspect, the present disclosure provides a method of isolating a recombinant adeno-associated virus (rAAV) genome using size exclusion chromatography. In some embodiments, the method includes subjecting a composition comprising rAAV particles to conditions in which the rAAV particles are denatured, and then subjecting the composition comprising denatured rAAV particles to size exclusion chromatography. In some embodiments, the mobile phase for size exclusion chromatography includes a salt, an organic solvent, or a detergent. In some embodiments, the mobile phase further includes a buffer. In some embodiments, the rAAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, or AAV. Contains capsid proteins of serotype HSC16. In some embodiments, the rAAV comprises a capsid protein of serotype AAV8 or AAV9.

In a further aspect, the disclosure provides a method of characterizing recombinant adeno-associated virus (rAAV) particles using size exclusion chromatography. Characterization of isolated rAAV particles includes, but is not limited to, determining the vector genome size purity of compositions containing isolated rAAV particles, folding or secondary folding of the vector genome within the capsid. and determining vector genome titer (Vg) of compositions containing isolated rAAV particles. In some embodiments, the mobile phase for size exclusion chromatography includes a salt, an organic solvent, or a detergent. In some embodiments, the mobile phase further includes a buffer. In some embodiments, the rAAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, or AAV. Contains capsid proteins of serotype HSC16. In some embodiments, the rAAV comprises a capsid protein of serotype AAV8 or AAV9.

In some embodiments, the methods disclosed herein are suitable for batch release, such as batch release testing and/or lot release testing. In some embodiments, the methods disclosed herein are performed as part of a lot release study.

In some embodiments, this disclosure provides:

[1. ] A method of producing recombinant virus particles, the method comprising:
a) providing a suspension cell culture of between about 200 liters and about 20,000 liters containing a population of cells capable of producing said recombinant virus particles;
b) combining one or more polynucleotides with at least one transfection reagent to form a first mixture, incubating said mixture to form a polynucleotide:transfection reagent complex; transferring the polynucleotide:transfection reagent complex to the suspension culture to transfect cells;
c) combining said one or more polynucleotides with said at least one transfection reagent to form a second mixture and incubating said mixture to form a polynucleotide:transfection reagent complex; , transferring the polynucleotide:transfection reagent complex to the suspension culture to transfect the cells;
d) maintaining said cell culture containing said transfected cells under conditions that allow production of said recombinant virus particles;
(i) said one or more polynucleotides include genes necessary for the production of recombinant virus particles;
(ii) the mixing, the incubating, and the transferring of steps b) and c) are each completed in less than about 60 minutes;
(iii) said transferring of step b) and step c) is carried out over a period of about 6 hours or less, and (iv) said transferring of step b) and step c) is performed simultaneously or sequentially in any order. carried out,
Said method.

[2. ] The method according to [1], wherein step c) is repeated one more time.

[3. ] The method according to [1], wherein step c) is repeated one or more times.

[4. ] The method according to [1], wherein step c) is repeated 1, 2, 3, 4, 5, 6, 7, or 8 times.

[5. [1] The combined volume of the polynucleotide:transfection reagent complex transferred to the suspension culture is between about 5% and about 20% of the volume of the suspension cell culture of step a). ] to [4].

[6. ] according to any one of [1] to [5], wherein said transferring of step c) is initiated between about 5 minutes and about 60 minutes after completing said transferring of step b). Method.

[7. ] The method of any one of [1] to [5], wherein said transferring of step c) is initiated within about 60 minutes of completing said transferring of step b).

[8. ] A method of increasing the production of recombinant virus particles, the method comprising:
a) providing a suspension cell culture of between about 200 liters and about 20,000 liters containing a population of cells capable of producing said recombinant virus particles;
b) combining one or more polynucleotides with at least one transfection reagent to form a first mixture, incubating said mixture to form a polynucleotide:transfection reagent complex; transferring the polynucleotide:transfection reagent complex to the suspension culture to transfect cells;
c) combining said one or more polynucleotides with said at least one transfection reagent to form a second mixture and incubating said mixture to form a polynucleotide:transfection reagent complex; , transferring the polynucleotide:transfection reagent complex to the suspension culture to transfect the cells;
d) maintaining said cell culture containing said transfected cells under conditions that allow production of said recombinant virus particles;
(i) said one or more polynucleotides include genes necessary for the production of recombinant viral particles;
(ii) the mixing, the incubating, and the transferring of steps b) and c) are each completed in less than about 60 minutes;
(iii) said transferring of step b) and step c) is carried out over a period of about 6 hours or less, and (iv) said transferring of step b) and step c) is performed simultaneously or sequentially in any order. carried out,
Said method.

[9. ] The method according to [8], wherein step c) is repeated one more time.

[10. ] The method according to [8], wherein step c) is repeated one or more times.

[11. ] The method according to [8], wherein step c) is repeated 1, 2, 3, 4, 5, 6, 7, or 8 times.

[12. [8] The combined volume of the polynucleotide:transfection reagent complex transferred to the suspension culture is between about 5% and about 20% of the volume of the suspension cell culture of step a). ] to [11].

[13. ] according to any one of [8] to [12], wherein said transferring of step c) is initiated between about 5 minutes and about 60 minutes after completing said transferring of step b). Method.

[14. ] The method of any one of [8] to [13], wherein the transferring of step c) is initiated within about 60 minutes of completing the transferring of step b).

[15. ] The method according to any one of [1] to [14], wherein mixing the one or more polynucleotides with at least one transfection reagent is performed by an in-line mixer.

[16. ] The method of any one of [1] to [15], wherein the mixing, the incubating, and the transferring of steps b) and c) are each completed in less than about 30 minutes. .

[17. ] The method of any one of [1] to [15], wherein the mixing, the incubating, and the transferring of steps b) and c) are each completed in less than about 35 minutes. .

[18. ] The method according to any one of [1] to [17], wherein the incubating steps of step b) and step c) are each for about 10 to about 20 minutes.

[19. ] The method according to any one of [1] to [17], wherein the incubating steps of step b) and step c) are each for about 10 to about 15 minutes.

[20. ] The method according to any one of [1] to [19], wherein the at least one transfection reagent comprises a stable cationic polymer.

[21. ] The method according to any one of [1] to [20], wherein the at least one transfection reagent comprises PEI.

[22. ] The method according to any one of [1] to [21], wherein the cell culture has a volume of between about 200 liters and about 5,000 liters.

[23. ] The method according to any one of [1] to [21], wherein the cell culture has a volume of between about 200 liters and about 2,000 liters.

[24. ] The method according to any one of [1] to [21], wherein the cell culture has a volume of between about 200 liters and about 1,000 liters.

[25. ] The method according to any one of [1] to [21], wherein the cell culture has a volume of between about 200 liters and about 500 liters.

[26. ] The cell culture is about 200 liters, about 300 liters, about 400 liters, about 500 liters, about 750 liters, about 1,000 liters, about 2,000 liters, about 3,000 liters, or about 5,000 liters. The method according to any one of [1] to [21], having a volume of liters.

[27. ] The method according to any one of [1] to [21], wherein the cell culture has a volume of about 500 liters.

[28. ] The method according to any one of [1] to [21], wherein the cell culture has a volume of about 1,000 liters.

[29. ] The method according to any one of [1] to [21], wherein the cell culture has a volume of about 2,000 liters.

[30. ] The method according to any one of [1] to [29], wherein the population of cells comprises a population of mammalian cells or insect cells.

[31. ] The method according to any one of [1] to [29], wherein the population of cells comprises a population of mammalian cells.

[21. ] The population of cells comprises a population of HEK293 cells, HEK-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, and/or PerC6 cells, [1] to [ 29].

[33. ] The method according to any one of [1] to [29], wherein the population of cells includes a population of HEK293 cells.

[34. ] The method according to any one of [1] to [33], wherein the suspension cell culture provided in step a) contains between about 2×10E+6 and about 10E+7 viable cells/ml.

[35. ] Conditions that allow said cell culture to produce said recombinant virus particles for about 2 days to about 10 days, about 3 days to about 5 days, or about 5 days to 14 days. The method according to any one of [1] to [34], wherein the method is maintained at

[36. ] The cell culture according to any one of [1] to [34] is maintained for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. Method.

[37. ] The method according to any one of [1] to [34], wherein the cell culture is maintained for at least about 3 days.

[38. ] The method according to any one of [1] to [34], wherein the cell culture is maintained for about 3 days.

[39. ] The method according to any one of [1] to [34], wherein the cell culture is maintained for about 4 days.

[40. ] The method according to any one of [1] to [39], wherein the recombinant virus particle is a recombinant adeno-associated virus (rAAV) particle or a recombinant lentivirus particle.

[41. ] The method according to any one of [1] to [39], wherein the recombinant virus particle is an rAAV particle.

[42. ] The rAAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, or AAV. The method according to [41], comprising a capsid protein of serotype HSC16.

[43. ] The rAAV particles are AAV8, AAV9, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, or AAV. The method according to [41], comprising a capsid protein of hu37 serotype.

[44. ] The method according to [41], wherein the rAAV particle comprises a capsid protein of AAV8 or AAV9 serotype.

[45. ] The method according to any one of [41] to [44], wherein the rAAV particle contains a genome containing a transgene.

[46. ] The method of [45], wherein the transgene comprises a regulatory element operably connected to a polynucleotide encoding a polypeptide.

[47. ] The method according to [46], wherein the regulatory element comprises one or more of an enhancer, a promoter, and a polyA region.

[48. ] The method according to [45] or [46], wherein the regulatory element and the polynucleotide encoding the polypeptide are heterologous.

[49. ] Any of [45] to [48], wherein the transgene encodes an antibody or antigen-binding fragment thereof, fusion protein, Fc fusion polypeptide, immunoadhesin, immunoglobulin, engineered protein, protein fragment, or enzyme. The method described in one.

[50. ] The transgene is an anti-VEGF Fab, iduronidase (IDUA), iduronate 2-sulfatase (IDS), low density lipoprotein receptor (LDLR), tripeptidyl peptidase 1 (TPP1), or VEGF receptor 1 (sFlt- The method according to any one of [45] to [48], which encodes the non-membrane-bound splice variant of 1).

[51. ] The transgene contains gamma-sarcoglycan, Rab escort protein 1 (REP1/CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide gated channel alpha 3 (CNGA3), cyclic nucleotide gated channel beta 3 (CNGB3), aromatic Family L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinosxin (RS1), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamate decarboxylase (GAD), glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, micro Dystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), arylsulfatase B ( ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha 1-antitrypsin (AAT) , phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neurturin (NRTN), neurotrophin-3 (NT-3/NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrial code NADH: ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dysferlin, MER proto-oncogene tyrosine kinase (MERTK), cyst The method according to any one of [45] to [48], which encodes a fibrotic transmembrane conductance regulator (CFTR) or a tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1) Fc fusion.

[52. ] said one or more polynucleotides,
a) rAAV genome to be packaged;
b) adenovirus helper functions necessary for packaging;
c) sufficient AAV rep protein for packaging; and
d) The method according to any one of [41] to [51], wherein the method encodes sufficient AAV cap protein for packaging.

[53. ] the one or more polynucleotides include a polynucleotide encoding the rAAV genome, a polynucleotide encoding the AAV rep protein and the AAV cap protein, and a polynucleotide encoding the adenovirus helper function; The method described in [52].

[54. ] The method according to any one of [52] or [53], wherein the adenovirus helper function includes at least one of the adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene. .

[55. ] The method according to any one of [412] to [54], further comprising collecting the rAAV particles.

[56. ] The method of any one of [41] to [55], wherein the cell culture produces between about 5×10e+10 GC/ml and about 1×10e+12 GC/ml of rAAV particles.

[57. ] the cell culture produces at least about twice as many rAAV particles, measured as GC/ml, than a reference method involving a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complexes. the method according to any one of [41] to [56].

[58. ] A composition comprising isolated rAAV particles produced by the method according to any one of [41] to [57].

Additional features and advantages of the compositions and methods described herein will become more apparent from the following detailed description when read in conjunction with the accompanying drawings.

Complex preparation process flow diagram for single dose 50L transient transfection. Complex preparation process flow diagram for single dose 200L transient transfection. Complex preparation process flow diagram for single dose 500L transient transfection. Bioreactor productivity of 50L, 200L, and 500L reactors using single-dose transfection. As the complexation time increases, the transfection complexes become larger. Complex preparation process flow diagram for testing the effect of complexation time on transient transfection efficiency. Effect of complexation time on transient transfection efficiency. Complex preparation process flow diagram for 200L split transient transfection. Productivity comparison between single-dose and split transfections in a 200l bioreactor. Robustness of split transfection. Complex preparation process flow diagram for 500L split transient transfection. Bioreactor productivity of a 500L reactor using split transfection. Split transfection process flow diagram for a 200L scale down of a 2,000L process.

DETAILED DESCRIPTION Described herein is a method for producing recombinant virus particles in large-scale culture, e.g., suspension culture, comprising polynucleotide:transfection reagent complexes for transfecting cells. transferring to the culture more than one volume of a separately produced composition comprising: wherein the transferring of the more than one volume of the composition is carried out over a period of about 6 hours or less; A method is provided in which the steps are performed simultaneously or sequentially. In some embodiments, the transfection reagent comprises a stable cationic polymer (eg, PEI). In some embodiments, the large-scale cell culture is between about 200 liters and about 20,000 liters and includes separately generated polynucleotide:transfection reagent complexes transferred to the culture. The combined volume of the composition is between about 5% and about 20% of the volume of the cell culture. In some embodiments, each volume of separately produced compositions comprising polynucleotide:transfection reagent complexes is produced in about 60 minutes or less (e.g., about 30 minutes or less) and transferred to cell culture. It will be done. In some embodiments, the combined volumes of separately produced compositions comprising the polynucleotide:transfection reagent complex are produced in a single batch in about 60 minutes or less (e.g., about 30 minutes or less); larger than the volume that can be transferred to large scale cultures. Without being bound to any particular theory, the methods disclosed herein allow for the transfer of large total volumes of compositions containing polynucleotide:transfection reagent complexes into cell cultures. provides increased productivity by providing increased productivity, where each component volume of the composition is produced and transferred to the cell culture in about 60 minutes or less (eg, about 30 minutes or less). In some embodiments, each volume of separately produced compositions comprising polynucleotide:transfection reagent complexes lasts for 60 minutes or less, 50 minutes or less, 40 minutes or less, 35 minutes or less, 30 minutes or less, 25 It is produced in minutes or less, or less than 20 minutes, and transferred to cell culture. In some embodiments, each volume of separately produced composition comprising a polynucleotide:transfection reagent complex is produced and transferred to cell culture in 30 minutes or less. In some embodiments, the productivity of the methods disclosed herein is greater than that of a reference method that involves transferring the same total volume of a composition comprising a polynucleotide:transfection reagent complex produced in a single batch. productivity is at least about twice as high as that of In some embodiments, productivity is quantified as virus particles per ml of culture at the time of harvest. In some embodiments, productivity is quantified as the number of virus particles recovered from a unit volume, eg, 1 ml of culture. In some embodiments, the cell culture is a suspension cell culture. In some embodiments, the cell culture comprises adherent cells that grow attached to microcarriers or macrocarriers in a stirred bioreactor. In some embodiments, the cell culture is a suspension cell culture comprising HEK293 cells.

In some embodiments, the recombinant virus particle is a recombinant adeno-associated virus (rAAV) particle. In some embodiments, the rAAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, or AAV. Contains capsid proteins of serotype HSC16. In some embodiments, the rAAV comprises a capsid protein of serotype AAV8 or AAV9.

Considering the extremely large number of rAAV particles required to prepare one therapeutic unit dose, a two-fold increase in rAAV yield significantly reduces the product cost per unit dose. Increased virus yields can correspondingly reduce the cost of consumables needed to produce rAAV particles, as well as the capital investment costs associated with constructing industrial virus purification facilities.

DEFINITIONS Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. To facilitate understanding of the methodologies of the present disclosure, several terms and expressions are defined below.

For example, modifying the amounts of components in the composition, concentrations of components in the composition, flow rates, rAAV particle yields, feed volumes, salt concentrations, and similar values, and ranges thereof, and the methods provided herein. As used in, "about" means, for example, due to typical measurement and handling procedures used to make the concentrate or use solution; due to accidental errors in these procedures; Means variations in numerical quantities that may result from differences in manufacture, sources, or purity of ingredients used in the practice; and similar considerations. The term "about" also encompasses the amount by which a composition or mixture having a particular initial concentration differs as it ages. The term "about" also encompasses amounts that vary by mixing or processing compositions or mixtures having a particular initial concentration. Whether modified by the term "about" or not, the claims include equivalents of that amount. In some embodiments, the term "about" means a range that is about 10-20% more or less than the number or range indicated. In a further embodiment, "about" means plus or minus 10% of the indicated number or range. For example, "about 10%" indicates a range of 9% to 11%.

"AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or a modification, derivative, or pseudotype thereof. The term encompasses all subtypes and both natural and recombinant forms, unless otherwise required. The abbreviation "rAAV" means recombinant adeno-associated virus. The term "AAV" refers to AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), 7 AAV type (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, and modifications thereof. , derivatives, or pseudotypes. "Primate AAV" refers to AAV that infects primates; "non-primate AAV" refers to AAV that infects non-primate mammals; and "bovine AAV" refers to AAV that infects bovine mammals. means, etc.

"Recombinant" as applied to AAV particles means that the AAV particles are the product of one or more procedures that result in an AAV particle construct that is essentially different from the AAV particle.

A recombinant adeno-associated virus particle "rAAV particle" is an encapsidated virus particle that contains at least one AAV capsid protein and a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, e.g., a transgene to be delivered to a mammalian cell). A viral particle composed of a polynucleotide rAAV vector genome. rAAV particles can be of any AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, or any of these), including any modifications, derivatives, or pseudotypes. derivatives/modifications/pseudotypes). Such AAV serotypes and derivatives/modifications/pseudotypes, as well as methods of generating such serotypes/derivatives/modifications/pseudotypes, are known in the art (e.g. Asokan et al. al., Mol. Ther. 20(4):699-708 (2012)).

The rAAV particles of the present disclosure include rAAV particles of any serotype, or any combination of serotypes (e.g., comprising two or more serotypes (e.g., comprising two or more of rAAV2, rAAV8, and rAAV9 particles)) particle population). In some embodiments, the rAAV particles are rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, or other rAAV particles, or a combination of two or more thereof. In some embodiments, the rAAV particles are rAAV8 or rAAV9 particles.

In some embodiments, the rAAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, or derivatives, modifications thereof. AAV capsid protein of a serotype selected from the group consisting of AAV capsid proteins, or pseudotypes. In some embodiments, the rAAV particle has an AAV capsid protein of serotype AAV8, AAV9, or a derivative, modification, or pseudotype thereof.

The term "cell culture" refers to cells grown in suspension or adherent in microcarriers or macrocarriers, bioreactors, roller bottles, hyperstacks, microspheres, macrospheres, flasks, etc., and supernatants or suspended cells. Refers to the components of the fluid itself, including, but not limited to, rAAV particles, cells, cell debris, cellular contaminants, colloidal particles, biomolecules, host cell proteins, nucleic acids, and lipids. , and flocculants. Large-scale approaches such as bioreactors, including suspension cultures and adherent cells grown attached to microcarriers or macrocarriers in stirred bioreactors, are encompassed by the term "cell culture." Cell culture procedures for both large-scale and small-scale production of viral particles or proteins are encompassed by this disclosure. In some embodiments, the term "cell culture" refers to cells grown in suspension. In some embodiments, the term "cell culture" refers to adherent cells grown attached to microcarriers or macrocarriers in a stirred bioreactor.

As used herein, "purifying", "purifying", "separating", "separating", "separating", "isolating", "isolating", or "isolating" The term is meant to increase the degree of purity of target products (eg, rAAV particles and rAAV genomes) from a sample containing the target product and one or more impurities. Typically, the degree of purity of the target product is increased by removing (completely or incompletely) at least one impurity from the sample. In some embodiments, the degree of purity of rAAV in a sample is determined by the removal (completely or incompletely) of one or more impurities from the sample by using the methods described herein. increases by

As used in this disclosure and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Whenever embodiments are described herein with the term "comprising," it also refers to another component that is described in terms of "consisting of" and/or "consisting essentially of." It should be understood that similar embodiments of are also provided. Also, whenever embodiments are described herein with the phrase "consisting essentially of", other similar embodiments are also described in terms of "consisting of". Please be understood as provided.

The term "and/or" as used herein in expressions such as "A and/or B" refers to both A and B, A or B, A (alone), and B (alone). intended to include. Similarly, the term "and/or" when used in expressions such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone) ).

When embodiments of the present disclosure are described with respect to a Markush group or other alternative group, the methods of the present disclosure encompass not only the recited group as a whole, but also each member of the group individually. It also includes all possible subgroups of the main group, as well as main groups that are missing one or more group members. The disclosed method also contemplates that any one or more of the group members in the disclosed method are explicitly excluded.

Methods for Recombinant Virus Production In some embodiments, the present disclosure provides a method for producing recombinant virus particles comprising:
a) providing a suspension cell culture of between about 200 liters and about 20,000 liters containing a population of cells capable of producing recombinant virus particles;
b) mixing one or more polynucleotides with at least one transfection reagent to form a first mixture, incubating the mixture to form a polynucleotide:transfection reagent complex, and adding the mixture to the cells. To transfect, transferring the polynucleotide:transfection reagent complex to a suspension culture;
c) combining one or more polynucleotides with at least one transfection reagent to form a second mixture, incubating the mixture to form a polynucleotide:transfection reagent complex, and adding the mixture to the cells. To transfect, transferring the polynucleotide:transfection reagent complex to a suspension culture;
d) maintaining a cell culture containing the transfected cells under conditions that allow production of recombinant virus particles;
Methods are provided in which the one or more polynucleotides include genes necessary for production of recombinant viral particles. In some embodiments, the one or more polynucleotides include one or more helper genes, rep genes, cap genes, and transgenes (eg, genes of interest or rAAV genomes to be packaged). In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to the suspension culture is between about 5% and about 20% of the volume of the cell culture in step a). In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to the suspension culture is between about 7% and about 15% of the volume of the cell culture in step a). In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to the suspension culture is about 10% of the volume of the cell culture in step a). In some embodiments, the transferring of step c) is initiated before completing the transferring of step b). In some embodiments, the transferring of step c) begins immediately after completing the transferring of step b). In some embodiments, transferring step c) is initiated between about 5 minutes and about 60 minutes after completing transferring step b). In some embodiments, transferring step c) is initiated within about 5 minutes of completing transferring step b). In some embodiments, transferring step c) is initiated within about 10 minutes of completing transferring step b). In some embodiments, transferring step c) is initiated within about 15 minutes of completing transferring step b). In some embodiments, transferring step c) is initiated within about 20 minutes of completing transferring step b). In some embodiments, transferring step c) is initiated within about 30 minutes of completing transferring step b). In some embodiments, transferring step c) is initiated within about 45 minutes of completing transferring step b). In some embodiments, transferring step c) is initiated within about 60 minutes of completing transferring step b). In some embodiments, the mixing, incubating, and transferring of steps b) and c) take less than about 90 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, about Completed in less than 35 minutes, less than about 30 minutes, less than about 25 minutes, or less than about 20 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 60 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 50 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 40 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 35 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 30 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 25 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 20 minutes. In some embodiments, the incubations of step b) and step c) are performed for between about 5 minutes and about 20 minutes, between about 10 minutes and about 20 minutes, between about 5 minutes and about 15 minutes, between about 10 minutes and about 15 minutes. or about 15 minutes to about 20 minutes. In some embodiments, the incubations of step b) and step c) occur for about 10 minutes to about 15 minutes. In some embodiments, incubating step b) and step c) for about 5 minutes, about 10 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, It lasts about 18 minutes, or about 20 minutes. In some embodiments, the incubations of step b) and step c) are performed for about 10 minutes. In some embodiments, the incubations of step b) and step c) are performed for about 12 minutes. In some embodiments, the incubations of step b) and step c) are performed for about 15 minutes. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 12 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 8 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 6 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 5 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 4 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 3 hours. In some embodiments, transferring step b) and step c) is performed over a period of about 12 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 9 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 8 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 7 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 6 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 5 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 5 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 3 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 2 hours or less. In some embodiments, transferring steps b) and c) are performed simultaneously or sequentially in any order. In some embodiments, transferring steps b) and c) are performed sequentially in any order. In some embodiments, step b) and step c) of mixing, incubating, and transferring are performed in the same manner. In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture comprises HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell culture has a volume between about 400 liters and about 5,000 liters. In some embodiments, the cell culture has a volume of about 500 liters. In some embodiments, the cell culture has a volume of about 2,000 liters. In some embodiments, the transfection reagent includes a cationic polymer. In some embodiments, the transfection reagent comprises PEI. In some embodiments, the recombinant virus particle is a recombinant adeno-associated virus (rAAV) particle. In some embodiments, the one or more polynucleotides include three polynucleotides, one encoding the cap and rep genes, and one encoding the adenovirus helper functions necessary for packaging (e.g., adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene), and one encoding the rAAV genome to be packaged). In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles are AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. PHB, and AAV. It has an AAV capsid protein of a serotype selected from the group consisting of 7m8. In some embodiments, the rAAV particles are AAV capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, and AAV. It has hu37.

In some embodiments, the present disclosure provides a method of producing recombinant viral particles comprising:
a) providing a suspension cell culture of between about 200 liters and about 20,000 liters containing a population of cells capable of producing recombinant virus particles;
b) mixing one or more polynucleotides with at least one transfection reagent to form a first mixture, incubating the mixture to form a polynucleotide:transfection reagent complex, and adding the mixture to the cells. To transfect, transferring the polynucleotide:transfection reagent complex to a suspension culture;
c) combining one or more polynucleotides with at least one transfection reagent to form a second mixture, incubating the mixture to form a polynucleotide:transfection reagent complex, and adding the mixture to the cells. To transfect, transferring the polynucleotide:transfection reagent complex to a suspension culture;
d) maintaining a cell culture containing the transfected cells under conditions that allow production of recombinant virus particles;
A method is provided, wherein the one or more polynucleotides contain genes necessary for the production of recombinant viral particles, and step c) is repeated one or more times. In some embodiments, step c) is repeated one more time. In some embodiments, step c) is repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, step c) is repeated twice. In some embodiments, step c) is repeated three times. In some embodiments, step c) is repeated four times. In some embodiments, step c) is repeated five times. In some embodiments, step c) is repeated six times. In some embodiments, step c) is repeated seven times. In some embodiments, step c) is repeated eight times. In some embodiments, step c) is repeated seven times. In some embodiments, step c) is repeated nine times. In some embodiments, step c) is repeated seven times. In some embodiments, step c) is repeated 10 times. In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to the suspension culture is between about 5% and about 20% of the volume of the cell culture in step a). In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to the suspension culture is between about 7% and about 15% of the volume of the cell culture in step a). In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to the suspension culture is about 10% of the volume of the cell culture in step a). In some embodiments, the transferring of step c) is initiated before completing the transferring of the preceding step. In some embodiments, the transferring of step c) is started immediately after completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated between about 5 minutes and about 60 minutes after completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 5 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 10 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 15 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 20 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 30 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 45 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 60 minutes of completing the transferring of the preceding step. In some embodiments, the mixing, incubating, and transferring of steps b) and c) take less than about 90 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, about Completed in less than 35 minutes, less than about 30 minutes, less than about 25 minutes, or less than about 20 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 60 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 50 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 40 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 35 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 30 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 25 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 20 minutes. In some embodiments, the incubations of step b) and step c) are performed for between about 5 minutes and about 20 minutes, between about 10 minutes and about 20 minutes, between about 5 minutes and about 15 minutes, between about 10 minutes and about 15 minutes. or about 15 minutes to about 20 minutes. In some embodiments, the incubations of step b) and step c) occur for about 10 minutes to about 15 minutes. In some embodiments, incubating step b) and step c) for about 5 minutes, about 10 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, It lasts about 18 minutes, or about 20 minutes. In some embodiments, the incubations of step b) and step c) are performed for about 10 minutes. In some embodiments, the incubations of step b) and step c) are performed for about 12 minutes. In some embodiments, the incubations of step b) and step c) are performed for about 15 minutes. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 12 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 8 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 6 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 5 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 4 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 3 hours. In some embodiments, transferring step b) and step c) is performed over a period of about 12 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 9 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 8 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 7 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 6 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 5 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 5 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 3 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 2 hours or less. In some embodiments, transferring steps b) and c) are performed simultaneously or sequentially in any order. In some embodiments, transferring steps b) and c) are performed sequentially in any order. In some embodiments, step b) and step c) of mixing, incubating, and transferring are performed in the same manner. In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture comprises HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell culture has a volume between about 400 liters and about 5,000 liters. In some embodiments, the cell culture has a volume of about 500 liters. In some embodiments, the cell culture has a volume of about 2,000 liters. In some embodiments, the transfection reagent includes a cationic polymer. In some embodiments, the one or more polynucleotides include one or more helper genes, rep genes, cap genes, and transgenes (eg, genes of interest). In some embodiments, the transfection reagent comprises PEI. In some embodiments, the recombinant virus particle is a recombinant adeno-associated virus (rAAV) particle. In some embodiments, the one or more polynucleotides include three polynucleotides (one encoding the cap and rep genes and one for adenovirus helper functions necessary for packaging (e.g., adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene), and one encoding the rAAV genome to be packaged). In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles are AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. PHB, and AAV. It has an AAV capsid protein of a serotype selected from the group consisting of 7m8. In some embodiments, the rAAV particles are AAV capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, and AAV. It has hu37.

In some embodiments, the present disclosure provides a method of increasing the production of recombinant viral particles, comprising:
a) providing a suspension cell culture of between about 200 liters and about 20,000 liters containing a population of cells capable of producing recombinant virus particles;
b) mixing one or more polynucleotides with at least one transfection reagent to form a first mixture, incubating the mixture to form a polynucleotide:transfection reagent complex, and adding the mixture to the cells. To transfect, transferring the polynucleotide:transfection reagent complex to a suspension culture;
c) combining one or more polynucleotides with at least one transfection reagent to form a second mixture, incubating the mixture to form a polynucleotide:transfection reagent complex, and adding the mixture to the cells. To transfect, transferring the polynucleotide:transfection reagent complex to a suspension culture;
d) maintaining a cell culture containing the transfected cells under conditions that allow production of recombinant virus particles;
Methods are provided in which the one or more polynucleotides include genes necessary for production of recombinant viral particles. In some embodiments, the method provides at least about twice as much polynucleotide:transfection reagent complex, measured as GC/ml, as a reference method that includes a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complex. generate rAAV particles. In some embodiments, the method increases rAAV production by at least about 50% compared to a reference method that includes a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complex. Increase by at least about 75%, or at least about 100%. In some embodiments, the methods disclosed herein improve rAAV production compared to reference methods that include a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complexes. at least about 2 times, at least about 3 times, or at least about 5 times. In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to the suspension culture is between about 5% and about 20% of the volume of the cell culture in step a). In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to the suspension culture is between about 7% and about 15% of the volume of the cell culture in step a). In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to the suspension culture is about 10% of the volume of the cell culture in step a). In some embodiments, the transferring of step c) is initiated before completing the transferring of step b). In some embodiments, the transferring of step c) begins immediately after completing the transferring of step b). In some embodiments, transferring step c) is initiated between about 5 minutes and about 60 minutes after completing transferring step b). In some embodiments, transferring step c) is initiated within about 5 minutes of completing transferring step b). In some embodiments, transferring step c) is initiated within about 10 minutes of completing transferring step b). In some embodiments, transferring step c) is initiated within about 15 minutes of completing transferring step b). In some embodiments, transferring step c) is initiated within about 20 minutes of completing transferring step b). In some embodiments, transferring step c) is initiated within about 30 minutes of completing transferring step b). In some embodiments, transferring step c) is initiated within about 45 minutes of completing transferring step b). In some embodiments, transferring step c) is initiated within about 60 minutes of completing transferring step b). In some embodiments, the mixing, incubating, and transferring of steps b) and c) take less than about 90 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, about Completed in less than 35 minutes, less than about 30 minutes, less than about 25 minutes, or less than about 20 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 60 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 50 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 40 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 35 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 30 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 25 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 20 minutes. In some embodiments, the incubations of step b) and step c) are performed for between about 5 minutes and about 20 minutes, between about 10 minutes and about 20 minutes, between about 5 minutes and about 15 minutes, between about 10 minutes and about 15 minutes. or about 15 minutes to about 20 minutes. In some embodiments, the incubations of step b) and step c) occur for about 10 minutes to about 15 minutes. In some embodiments, incubating step b) and step c) for about 5 minutes, about 10 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, It lasts about 18 minutes, or about 20 minutes. In some embodiments, the incubations of step b) and step c) are performed for about 10 minutes. In some embodiments, the incubations of step b) and step c) are performed for about 12 minutes. In some embodiments, the incubations of step b) and step c) are performed for about 15 minutes. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 12 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 8 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 6 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 5 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 4 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 3 hours. In some embodiments, transferring step b) and step c) is performed over a period of about 12 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 9 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 8 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 7 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 6 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 5 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 5 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 3 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 2 hours or less. In some embodiments, transferring steps b) and c) are performed simultaneously or sequentially in any order. In some embodiments, transferring steps b) and c) are performed sequentially in any order. In some embodiments, step b) and step c) of mixing, incubating, and transferring are performed in the same manner. In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture comprises HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell culture has a volume between about 400 liters and about 5,000 liters. In some embodiments, the cell culture has a volume of about 500 liters. In some embodiments, the cell culture has a volume of about 2,000 liters. In some embodiments, the one or more polynucleotides include one or more helper genes, rep genes, cap genes, and transgenes (eg, genes of interest). In some embodiments, the transfection reagent includes a cationic polymer. In some embodiments, the transfection reagent comprises PEI. In some embodiments, the recombinant virus particle is a recombinant adeno-associated virus (rAAV) particle. In some embodiments, the one or more polynucleotides include three polynucleotides, one encoding the cap and rep genes, and one encoding the adenovirus helper functions necessary for packaging (e.g., adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene), and one encoding the rAAV genome to be packaged). In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles are AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. PHB, and AAV. It has an AAV capsid protein of a serotype selected from the group consisting of 7m8. In some embodiments, the rAAV particles are AAV capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, and AAV. It has hu37.

In some embodiments, the present disclosure provides a method of increasing the production of recombinant viral particles, comprising:
a) providing a suspension cell culture of between about 200 liters and about 20,000 liters containing a population of cells capable of producing recombinant virus particles;
b) mixing one or more polynucleotides with at least one transfection reagent to form a first mixture, incubating the mixture to form a polynucleotide:transfection reagent complex, and adding the mixture to the cells. To transfect, transferring the polynucleotide:transfection reagent complex to a suspension culture;
c) combining one or more polynucleotides with at least one transfection reagent to form a second mixture, incubating the mixture to form a polynucleotide:transfection reagent complex, and adding the mixture to the cells. To transfect, transferring the polynucleotide:transfection reagent complex to a suspension culture;
d) maintaining a cell culture containing the transfected cells under conditions that allow production of recombinant virus particles;
A method is provided, wherein the one or more polynucleotides contain genes necessary for the production of recombinant viral particles, and step c) is repeated one or more times. In some embodiments, the method provides at least about twice as much polynucleotide:transfection reagent complex, measured as GC/ml, as a reference method that includes a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complex. generate rAAV particles. In some embodiments, the method increases rAAV production by at least about 50% compared to a reference method that includes a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complex. Increase by at least about 75%, or at least about 100%. In some embodiments, the methods disclosed herein improve rAAV production compared to reference methods that include a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complexes. at least about 2 times, at least about 3 times, or at least about 5 times. In some embodiments, step c) is repeated one more time. In some embodiments, step c) is repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, step c) is repeated twice. In some embodiments, step c) is repeated three times. In some embodiments, step c) is repeated four times. In some embodiments, step c) is repeated five times. In some embodiments, step c) is repeated six times. In some embodiments, step c) is repeated seven times. In some embodiments, step c) is repeated eight times. In some embodiments, step c) is repeated seven times. In some embodiments, step c) is repeated nine times. In some embodiments, step c) is repeated seven times. In some embodiments, step c) is repeated 10 times. In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to the suspension culture is between about 5% and about 20% of the volume of the cell culture in step a). In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to the suspension culture is between about 7% and about 15% of the volume of the cell culture in step a). In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to the suspension culture is about 10% of the volume of the cell culture in step a). In some embodiments, the transferring of step c) is initiated before completing the transferring of the preceding step. In some embodiments, the transferring of step c) is started immediately after completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated between about 5 minutes and about 60 minutes after completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 5 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 10 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 15 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 20 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 30 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 45 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 60 minutes of completing the transferring of the preceding step. In some embodiments, the mixing, incubating, and transferring of steps b) and c) take less than about 90 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, about Completed in less than 35 minutes, less than about 30 minutes, less than about 25 minutes, or less than about 20 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 60 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 50 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 40 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 35 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 30 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 25 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 20 minutes. In some embodiments, the incubations of step b) and step c) are performed for between about 5 minutes and about 20 minutes, between about 10 minutes and about 20 minutes, between about 5 minutes and about 15 minutes, between about 10 minutes and about 15 minutes. or about 15 minutes to about 20 minutes. In some embodiments, the incubations of step b) and step c) occur for about 10 minutes to about 15 minutes. In some embodiments, incubating step b) and step c) for about 5 minutes, about 10 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, It lasts about 18 minutes, or about 20 minutes. In some embodiments, the incubations of step b) and step c) are performed for about 10 minutes. In some embodiments, the incubations of step b) and step c) are performed for about 12 minutes. In some embodiments, the incubations of step b) and step c) are performed for about 15 minutes. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 12 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 8 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 6 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 5 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 4 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 3 hours. In some embodiments, transferring step b) and step c) is performed over a period of about 12 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 9 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 8 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 7 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 6 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 5 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 5 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 3 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 2 hours or less. In some embodiments, transferring steps b) and c) are performed simultaneously or sequentially in any order. In some embodiments, transferring steps b) and c) are performed sequentially in any order. In some embodiments, step b) and step c) of mixing, incubating, and transferring are performed in the same manner. In some embodiments, the cell culture is a suspension culture. In some embodiments, the cell culture comprises HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell culture has a volume between about 400 liters and about 5,000 liters. In some embodiments, the cell culture has a volume of about 500 liters. In some embodiments, the cell culture has a volume of about 2,000 liters. In some embodiments, the transfection reagent includes a cationic polymer. In some embodiments, the transfection reagent comprises PEI. In some embodiments, the recombinant virus particle is a recombinant adeno-associated virus (rAAV) particle. In some embodiments, the one or more polynucleotides include one or more helper genes, rep genes, cap genes, and transgenes (eg, genes of interest or rAAV genomes to be packaged). In some embodiments, the one or more polynucleotides include three polynucleotides, one encoding the cap and rep genes, and one encoding the adenovirus helper functions necessary for packaging (e.g., adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene), and one encoding the rAAV genome to be packaged). In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles are AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. PHB, and AAV. It has an AAV capsid protein of a serotype selected from the group consisting of 7m8. In some embodiments, the rAAV particles are AAV capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, and AAV. It has hu37.

In some embodiments, the present disclosure provides a method of producing recombinant adeno-associated virus (rAAV) particles comprising:
a) providing a suspension cell culture of between about 200 liters and about 20,000 liters containing a population of cells capable of producing rAAV;
b) mixing one or more polynucleotides with at least one transfection reagent to form a first mixture, incubating the mixture to form a polynucleotide:transfection reagent complex, and adding the mixture to the cells. To transfect, transferring the polynucleotide:transfection reagent complex to a suspension cell culture;
c) combining one or more polynucleotides with at least one transfection reagent to form a second mixture, incubating the mixture to form a polynucleotide:transfection reagent complex, and adding the mixture to the cells. To transfect, transferring the polynucleotide:transfection reagent complex to a suspension cell culture;
d) maintaining a suspension cell culture containing the transfected cells under conditions that allow the production of rAAV particles;
A method is provided in which the transfection reagent includes PEI and the one or more polynucleotides include genes necessary for production of rAAV particles. In some embodiments, the present disclosure provides a method of increasing the production of recombinant adeno-associated virus (rAAV) particles, comprising:
a) providing a suspension cell culture of between about 200 liters and about 20,000 liters containing a population of cells capable of producing rAAV particles;
b) mixing one or more polynucleotides with at least one transfection reagent to form a first mixture, incubating the mixture to form a polynucleotide:transfection reagent complex, and adding the mixture to the cells. To transfect, transferring the polynucleotide:transfection reagent complex to a suspension culture;
c) combining one or more polynucleotides with at least one transfection reagent to form a second mixture, incubating the mixture to form a polynucleotide:transfection reagent complex, and adding the mixture to the cells. To transfect, transferring the polynucleotide:transfection reagent complex to a suspension culture;
d) maintaining a suspension cell culture containing transfected cells under conditions that allow the production of rAAV particles, wherein the transfection reagent comprises PEI and the one or more polynucleotides are present in the rAAV particles. Methods are provided, including genes necessary for production. In some embodiments, the method provides at least about twice as much polynucleotide:transfection reagent complex, measured as GC/ml, as a reference method that includes a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complex. generate rAAV particles. In some embodiments, the method increases rAAV production by at least about 50% compared to a reference method that includes a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complex. Increase by at least about 75%, or at least about 100%. In some embodiments, the methods disclosed herein improve rAAV production compared to reference methods that include a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complexes. at least about 2 times, at least about 3 times, or at least about 5 times. In some embodiments, the present disclosure provides a method of producing recombinant adeno-associated virus (rAAV) particles, comprising: a) about 200 liters to about 20,000 liters comprising a population of cells capable of producing rAAV particles; b) combining one or more polynucleotides with at least one transfection reagent to form a first mixture, a polynucleotide:transfection reagent complex; c) mixing one or more polynucleotides with at least one transfection reagent to form a second mixture, a polynucleotide:transfection reagent complex; d) incubating the mixture to form a polynucleotide:transfection reagent complex to a culture to transfect the cells; d) a suspension cell culture containing the transfected cells; the transfection reagent comprises a PEI, the one or more polynucleotides comprise a gene necessary for the generation of rAAV particles, and step c) is carried out once. A method is provided which is repeated above. In some embodiments, step c) is repeated one more time. In some embodiments, step c) is repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, step c) is repeated twice. In some embodiments, step c) is repeated three times. In some embodiments, step c) is repeated four times. In some embodiments, step c) is repeated five times. In some embodiments, step c) is repeated six times. In some embodiments, step c) is repeated seven times. In some embodiments, step c) is repeated eight times. In some embodiments, step c) is repeated seven times. In some embodiments, step c) is repeated nine times. In some embodiments, step c) is repeated seven times. In some embodiments, step c) is repeated 10 times. In some embodiments, the present disclosure provides a method of increasing the production of recombinant adeno-associated virus (rAAV) particles, comprising: a) about 200 liters containing a population of cells capable of producing rAAV particles; 000 liters of suspension cell culture; b) combining one or more polynucleotides with at least one transfection reagent to form a first mixture; polynucleotide:transfection reagent; incubating the mixture to form a complex; c) combining the one or more polynucleotides with at least one transfection reagent to form a second mixture; polynucleotide:transfection reagent; incubating the mixture to form the complex and transferring the polynucleotide:transfection reagent complex to a culture to transfect the cells; and d) a suspension cell culture containing the transfected cells. the transfection reagent comprises a PEI, the one or more polynucleotides comprise a gene necessary for the production of rAAV particles, and step c) A method is provided that is repeated one or more times. In some embodiments, the method provides at least about twice as much polynucleotide:transfection reagent complex, measured as GC/ml, as a reference method that includes a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complex. generate rAAV particles. In some embodiments, the method increases rAAV production by at least about 50% compared to a reference method that includes a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complex. Increase by at least about 75%, or at least about 100%. In some embodiments, the methods disclosed herein improve rAAV production compared to reference methods that include a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complexes. at least about 2 times, at least about 3 times, or at least about 5 times. In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to the suspension cell culture is between about 5% and about 20% of the volume of the suspension cell culture in step a). be. In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to the suspension cell culture is between about 5% and about 20% of the volume of the suspension cell culture in step a). be. In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to the suspension cell culture is between about 7% and about 15% of the volume of the suspension cell culture in step a). be. In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to the suspension cell culture is about 10% of the volume of the suspension cell culture in step a). In some embodiments, the transferring of step c) is initiated before completing the transferring of the preceding step. In some embodiments, the transferring of step c) is started immediately after completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated between about 5 minutes and about 60 minutes after completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 5 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 10 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 15 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 20 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 30 minutes of completing the transferring of the preceding step. In some embodiments, the transferring of step c) is initiated within about 45 minutes of completing the transferring of the preceding step. In some embodiments, transferring step c) is initiated within about 60 minutes of completing transferring step b). In some embodiments, the mixing, incubating, and transferring of steps b) and c) take less than about 90 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, about Completed in less than 35 minutes, less than about 30 minutes, less than about 25 minutes, or less than about 20 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 60 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 50 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 40 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 35 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 30 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 25 minutes. In some embodiments, the mixing, incubating, and transferring steps b) and c) are each completed in less than about 20 minutes. In some embodiments, the incubations of step b) and step c) are performed for between about 5 minutes and about 20 minutes, between about 10 minutes and about 20 minutes, between about 5 minutes and about 15 minutes, between about 10 minutes and about 15 minutes. or about 15 minutes to about 20 minutes. In some embodiments, the incubations of step b) and step c) occur for about 10 minutes to about 15 minutes. In some embodiments, incubating step b) and step c) for about 5 minutes, about 10 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, It lasts about 18 minutes, or about 20 minutes. In some embodiments, the incubations of step b) and step c) are performed for about 10 minutes. In some embodiments, the incubations of step b) and step c) are performed for about 12 minutes. In some embodiments, the incubations of step b) and step c) are performed for about 15 minutes. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 12 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 8 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 6 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 5 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 4 hours. In some embodiments, transferring steps b) and c) are performed over a period of between about 1 hour and about 3 hours. In some embodiments, transferring step b) and step c) is performed over a period of about 12 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 9 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 8 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 7 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 6 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 5 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 5 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 3 hours or less. In some embodiments, transferring step b) and step c) is performed over a period of about 2 hours or less. In some embodiments, transferring steps b) and c) are performed simultaneously or sequentially in any order.
In some embodiments, transferring steps b) and c) are performed sequentially in any order. In some embodiments, step b) and step c) of mixing, incubating, and transferring are performed in the same manner. In some embodiments, the suspension cell culture comprises HEK293 cells adapted for growth in suspension cell culture. In some embodiments, the suspension cell culture has a volume between about 400 liters and about 10,000 liters. In some embodiments, the cell culture has a volume of about 500 liters. In some embodiments, the cell culture has a volume of about 2,000 liters. In some embodiments, the one or more polynucleotides include one or more helper genes, rep genes, cap genes, and transgenes (eg, genes of interest or rAAV genomes to be packaged). In some embodiments, the one or more polynucleotides include three polynucleotides, one encoding the cap and rep genes, and one encoding the adenovirus helper functions necessary for packaging (e.g., adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene), and one encoding the rAAV genome to be packaged). In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles are AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. PHB, and AAV. It has an AAV capsid protein of a serotype selected from the group consisting of 7m8. In some embodiments, the rAAV particles are AAV capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, and AAV. It has hu37.

Transfection-based recombinant virus particle production systems are known to those skilled in the art. For example, Reiser et al. , Gene Ther 7(11):910-3 (2000); Dull et al. , J Virol. 72(11):8463-8471 (1998); Hoffmann et al. , PNAS 97(11) 6108-6113 (2000); Milian et al. , Vaccine 35(26):3423-3430 (2017), each of which is incorporated by reference in its entirety. The methods disclosed herein can be used to produce recombinant virus particles in a transfection-based production system. In some embodiments, the recombinant viral particles are recombinant dengue virus, recombinant Ebola virus, recombinant human papillomavirus (HPV), recombinant human immunodeficiency virus (HIV), recombinant adeno-associated virus (AAV), recombinant Recombinant lentivirus, recombinant influenza virus, recombinant vesicular stomatitis virus (VSV), recombinant poliovirus, recombinant adenovirus, recombinant retrovirus, recombinant vaccinia, recombinant reovirus, recombinant measles, recombinant virus Castle disease virus (NDV), recombinant herpes zoster virus (HZV), recombinant herpes simplex virus (HSV), or recombinant baculovirus. In some embodiments, the recombinant virus particle is a recombinant adeno-associated virus (AAV), a recombinant lentivirus, or a recombinant influenza virus. In some embodiments, the recombinant viral particle is a recombinant lentivirus. In some embodiments, the recombinant virus particle is a recombinant influenza virus. In some embodiments, the recombinant virus particle is a recombinant baculovirus. In some embodiments, the recombinant viral particle is a recombinant adeno-associated virus (AAV). In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles are AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. PHB, and AAV. It has an AAV capsid protein of a serotype selected from the group consisting of 7m8. In some embodiments, the rAAV particles are AAV capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, and AAV. It has hu37.

Any suitable transfection reagent for transfecting cells known in the art can be used to generate recombinant virus particles (e.g., rAAV particles) according to the methods disclosed herein. can. In some embodiments, the cells are HEK293 cells (eg, HEK293 cells adapted to suspension culture). In some embodiments, the methods disclosed herein include transfecting cells using chemical-based transfection methods. In some embodiments, the methods disclosed herein include transfecting cells with a cationic organic carrier. For example, Gigante et al. , MedChemComm 10(10):1692-1718 (2019); Damen et al. See MedChemComm 9(9):1404-1425 (2018), each of which is incorporated by reference in its entirety. In some embodiments, the cationic organic carrier comprises a lipid, such as DOTMA, DOTAP, helper lipids (Dope, cholesterol), and combinations thereof. In some embodiments, the cationic organic carrier comprises polyvalent cationic lipids, such as DOSPA, DOGS, and mixtures thereof. In some embodiments, the cationic organic carrier comprises a dipolar lipid, or a bolaamphiphile (bola). In some embodiments, the cationic organic carrier comprises a biologically reducible and/or dimerizable lipid. In some embodiments, the cationic organic carrier comprises a gemini surfactant. In some embodiments, the cationic organic carrier comprises Lipofectin(TM), Transfectam(TM), Lipofectamine(TM), Lipofectamine 2000(TM), or Lipofectamine PLUS 2000(TM). In some embodiments, the cationic organic carrier is a polymer, such as poly(L-lysine) (PLL), polyethyleneimine (PEI), polysaccharides (chitosan, dextran, cyclodextrin (CD)), poly[2- (dimethylamino)ethyl methacrylate] (PDMAEMA), and dendrimers (polyamidoamine (PAMAM), poly(propylene imine) (PPI)). In some embodiments, the cationic organic carrier is a peptide, such as a basic amino acid rich peptide ( _CWL18 ), a cell penetrating peptide (CPP) (Arg rich peptide (octaarginine, TAT)), localization signal (NLS) (SV40), and targeting (RGD). In some embodiments, the cationic organic carrier comprises a polymer (eg, PEI) in combination with a cationic liposome. Paris et al. , Molecules 25(14):3277 (2020) (incorporated herein by reference in its entirety). In some embodiments, transfection reagents include calcium phosphate, highly branched organic compounds (dendrimers), cationic polymers (eg, DEAE dextran or polyethyleneimine (PEI)), lipofection.

In some embodiments, the transfection reagent is poly(L-lysine) (PLL), polyethyleneimine (PEI), linear PEI, branched PEI, dextran, cyclodextrin (CD), poly[2- (dimethylamino)ethyl methacrylate] (PDMAEMA), polyamidoamine (PAMAM), poly(propyleneimine) (PPI), or mixtures thereof. In some embodiments, the transfection reagent comprises polyethyleneimine (PEI), linear PEI, branched PEI, or mixtures thereof. In some embodiments, the transfection reagent comprises polyethyleneimine (PEI). In some embodiments, the transfection reagent comprises linear PEI. In some embodiments, the transfection reagent comprises a branched PEI. In some embodiments, the transfection reagent comprises polyethyleneimine (PEI) having a molecular weight between about 5 and about 25 kDa. In some embodiments, the transfection reagent comprises PEGylated polyethyleneimine (PEI). In some embodiments, the transfection reagent comprises a modified polyethyleneimine (PEI) with attached hydrophobic moieties (eg, cholesterol, choline, alkyl groups, and some amino acids).

Composition polynucleotide:transfection reagent complexes can be prepared by any method known to those skilled in the art. In some embodiments, combining the one or more polynucleotides with at least one transfection reagent comprises diluting each of the transfection reagent and the one or more polynucleotides in a sterile liquid (e.g., tissue culture medium). and mixing the diluted transfection reagent and the diluted one or more polynucleotides. In some embodiments, mixing includes transferring the diluted one or more polynucleotides and the diluted at least one transfection reagent from two separate containers to a new container. In some embodiments, transferring the diluted one or more polynucleotides and the diluted at least one transfection reagent to a new container is about 500 ml/min, about 1 liter/min, about 2 liters/minute 3 liters/min, 4 liters/min, 5 liters/min, 6 liters/min, 7 liters/min, 8 liters/min, 9 liters/min, or 10 liters/min is carried out at a speed of In some embodiments, transferring is performed at a rate of about 3 liters/minute. In some embodiments, transferring is performed at a rate of about 4 liters/minute. In some embodiments, transferring is performed at a rate of about 5 liters/minute. In some embodiments, transferring is performed at a rate of about 6 liters/minute. In some embodiments, mixing the diluted one or more polynucleotides with the diluted at least one transfection reagent is performed by an in-line mixer. In some embodiments, the in-line mixer is a low shear in-line mixer. In some embodiments, the in-line mixer is a static in-line mixer. Inline mixers suitable for mixture of polynucleotide and trait transfer reagent are known in our technical field, for example, SARTORIUS (FLEXSAFE (registered trademark) PRO MIXER), Analytical Scientific INSTRUMENTS US C (HyperShear (trademark)), fluitec , or available from STRIKO. Those skilled in the art will understand that dilution and mixing are performed to produce a composition containing the transfection reagent and polynucleotide in the desired ratio and concentration. In some embodiments, diluting and mixing at least one transfection reagent and one or more polynucleotides is a composition comprising the transfection reagent and polynucleotide in a weight ratio of between about 1:5 and 5:1. generate. In some embodiments, the weight ratio of transfection reagent and polynucleotide is between about 1:3 and 3:1. In some embodiments, the weight ratio of transfection reagent and polynucleotide is between about 1:3 and 1:1. In some embodiments, the weight ratio of transfection reagent and polynucleotide is between about 1:2 and 1:1.5. In some embodiments, the weight ratio of transfection reagent and polynucleotide is about 1:5, 1:4, 1:3, 1:2.5, 1:2, 1:1.75, 1:1 .5, 1:1.25, 1:1, 1.25:1, 1.5:1, 1.75:1, 2:1, 2.5:1, 3:1, 4:1, or The ratio is 5:1. In some embodiments, the weight ratio of transfection reagent and polynucleotide is about 1:2. In some embodiments, the weight ratio of transfection reagent and polynucleotide is about 1:1.75. In some embodiments, the weight ratio of transfection reagent and polynucleotide is about 1:1.5. In some embodiments, the weight ratio of transfection reagent and polynucleotide is about 1:1.25. In some embodiments, the weight ratio of transfection reagent and polynucleotide is about 1:1. In some embodiments, the weight ratio of transfection reagent and polynucleotide is about 1.25:1. In some embodiments, the weight ratio of transfection reagent and polynucleotide is about 1.5:1. In some embodiments, the weight ratio of transfection reagent and polynucleotide is about 1.75:1. In some embodiments, the weight ratio of transfection reagent and polynucleotide is about 2:1. In some embodiments, the composition comprises between about 1 μg and about 20 μg of one or more polynucleotides. In some embodiments, the one or more polynucleotides include three plasmids. In some embodiments, the one or more polynucleotides include two plasmids. In some embodiments, the one or more polynucleotides comprises one plasmid. In some embodiments, the recombinant virus is recombinant AAV and the one or more polynucleotides include three polynucleotides, one encoding the cap and rep genes and one necessary for packaging. adenovirus helper functions (eg, adenovirus E1a, E1b, E4, E2a, and VA genes, and one encoding the rAAV genome to be packaged). In some embodiments, the rAAV particles are AAV8 or AAV9 particles. In some embodiments, the rAAV particles are AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. PHB, and AAV. It has an AAV capsid protein of a serotype selected from the group consisting of 7m8. In some embodiments, the rAAV particles are AAV capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, and AAV. It has hu37. In some embodiments, the transfection reagent is PEI.

In some embodiments, a composition comprising a transfection reagent and one or more polynucleotides is incubated to allow formation of a polynucleotide:transfection reagent complex. In some embodiments, incubation occurs at room temperature. In some embodiments, incubating comprises shaking the composition, for example, in a shaker between about 100 rpm and about 200 rpm. In some embodiments, incubating is for about 5 minutes to about 20 minutes, for about 10 minutes to about 20 minutes, for about 5 minutes to about 15 minutes, for about 10 minutes to about 15 minutes, or for about 15 minutes to It takes about 20 minutes. In some embodiments, incubation occurs for about 5 minutes to about 20 minutes. In some embodiments, incubation occurs for about 10 minutes to about 15 minutes. In some embodiments, incubating for about 5 minutes, about 10 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, or about 20 minutes. It will be done. In some embodiments, incubation is for about 11 minutes. In some embodiments, incubation is for about 12 minutes. In some embodiments, incubation is for about 13 minutes. In some embodiments, incubation is for about 14 minutes. In some embodiments, incubation is for about 15 minutes. In some embodiments, incubation is performed for 15 minutes or less. In some embodiments, incubation is performed for 10 minutes or less. In some embodiments, incubating is for about 5 minutes, about 10 minutes, or about 15 minutes. In some embodiments, incubation is for about 10 minutes. In some embodiments, the length of the incubation is less than about 90 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, less than about 35 minutes of mixing, incubating, and transferring. , less than about 30 minutes, less than about 25 minutes, or less than about 20 minutes. In some embodiments, mixing, incubating, and transferring is completed in less than about 30 minutes. In some embodiments, the one or more polynucleotides include genes necessary for production of recombinant AAV particles. In some embodiments, the transfection reagent comprises PEI.

Methods for transferring polynucleotide:transfection reagent complexes to suspension cultures are known to those skilled in the art. In some embodiments, transferring is performed using a peristaltic pump. In some embodiments, transferring is performed at a rate between about 100 ml/min and about 10 liters/min. In some embodiments, transferring is about 500 ml/min, about 1 liter/min, about 2 liters/min, about 3 liters/min, about 4 liters/min, about 5 liters/min, about 6 liters/min. minutes, about 7 liters/minute, about 8 liters/minute, about 9 liters/minute, or about 10 liters/minute. In some embodiments, transferring is performed at a rate of about 3 liters/minute. In some embodiments, transferring is performed at a rate of about 4 liters/minute. In some embodiments, transferring is performed at a rate of about 5 liters/minute. In some embodiments, transferring is performed at a rate of about 6 liters/minute. In some embodiments, transferring is performed at a rate of about 7 liters/minute. In some embodiments, transferring is performed at a rate of about 8 liters/minute. In some embodiments, transferring is performed at a rate of about 9 liters/minute. In some embodiments, transferring is performed at a rate of about 10 liters/minute. In some embodiments, the rate of transfer is such that mixing, incubating, and transferring takes less than about 90 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, less than about 35 minutes, about Set to complete in less than 30 minutes, less than about 25 minutes, or less than about 20 minutes. In some embodiments, mixing, incubating, and transferring is completed in less than about 30 minutes. In some embodiments, mixing, incubating, and transferring is completed in less than about 35 minutes. In some embodiments, the polynucleotide includes genes necessary for production of recombinant AAV particles. In some embodiments, the transfection reagent comprises PEI. In some embodiments, the culture comprises HEK293 cells (eg, HEK293 cells adapted to suspension culture).

In some embodiments, separately generated polynucleotide:transfection reagent complexes are generated using the same process. In some embodiments, one or more polynucleotides are separately mixed with at least one transfection reagent to form a first mixture, and to form a polynucleotide:transfection reagent complex, The same process is used to incubate the mixture and transfer the polynucleotide:transfection reagent complex to a suspension culture to transfect cells. In some embodiments, separately transferring the polynucleotide:transfection reagent complexes to the suspension culture comprises transferring the same volume of polynucleotide:transfection reagent complexes. In some embodiments, separately transferring polynucleotide:transfection reagent complexes to a suspension culture comprises transferring different volumes of polynucleotide:transfection reagent complexes. In some embodiments, the one or more polynucleotides include genes necessary for production of recombinant AAV particles. In some embodiments, the transfection reagent comprises PEI. In some embodiments, the culture comprises HEK293 cells (eg, HEK293 cells adapted to suspension culture).

In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to a cell culture is a cell culture containing a population of cells capable of producing recombinant viral particles (e.g., rAAV particles). between about 5% and about 20% of the volume. In some embodiments, the combined volume of transferred polynucleotide:transfection reagent complex is between about 7% and about 15% of the volume of the cell culture. In some embodiments, the combined volume of transferred polynucleotide:transfection reagent complex is about 10% of the volume of the cell culture. In some embodiments, the one or more polynucleotides include genes necessary for production of recombinant AAV particles. In some embodiments, the transfection reagent comprises PEI. In some embodiments, the culture comprises HEK293 cells (eg, HEK293 cells adapted to suspension culture).

In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to cell culture ranges from about 0.1 μg of one or more polynucleotides/10E+6 live cells/ml to about 5 μg of 1 1 or more polynucleotides/10E+6 live cells/ml. In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to cell culture ranges from about 0.2 μg of one or more polynucleotides/10E+6 live cells/ml to about 2 μg of 1 1 or more polynucleotides/10E+6 live cells/ml. In some embodiments, the combined volume of polynucleotide:transfection reagent complex transferred to cell culture is about 0.2, 0.3, 0.4, 0.5, 0.6, 0 .7, 0.8, 0.9, or 1 μg of one or more polynucleotides/10E+6 live cells/ml. In some embodiments, the one or more polynucleotides include genes necessary for production of recombinant AAV particles. In some embodiments, the transfection reagent comprises PEI. In some embodiments, the culture comprises HEK293 cells (eg, HEK293 cells adapted to suspension culture).

In some embodiments, the cell culture has a volume between about 400 liters and about 20,000 liters. In some embodiments, the cell culture has a volume between about 500 liters and about 20,000 liters. In some embodiments, the cell culture has a volume between about 700 liters and about 20,000 liters. In some embodiments, the cell culture has a volume between about 1,000 liters and about 20,000 liters. In some embodiments, the cell culture has a volume between about 400 liters and about 10,000 liters. In some embodiments, the cell culture has a volume between about 500 liters and about 10,000 liters. In some embodiments, the cell culture has a volume between about 700 liters and about 10,000 liters. In some embodiments, the cell culture has a volume between about 1,000 liters and about 10,000 liters. In some embodiments, the cell culture has a volume between about 400 liters and about 5,000 liters. In some embodiments, the cell culture has a volume between about 500 liters and about 5,000 liters. In some embodiments, the cell culture has a volume between about 700 liters and about 5,000 liters. In some embodiments, the cell culture has a volume between about 1,000 liters and about 5,000 liters. In some embodiments, the cell culture volume referred to herein is the final bioreactor/vessel volume as listed in Table 1. In some embodiments, the culture comprises HEK293 cells (eg, HEK293 cells adapted to suspension culture).

In some embodiments, the cell culture has a volume between about 200 liters and about 5,000 liters. In some embodiments, the cell culture has a volume between about 200 liters and about 2,000 liters. In some embodiments, the cell culture has a volume between about 200 liters and about 1,000 liters. In some embodiments, the cell culture has a volume between about 200 liters and about 500 liters. In some embodiments, the cell culture volume referred to herein is the final bioreactor/vessel volume as listed in Table 1. In some embodiments, the culture comprises HEK293 cells (eg, HEK293 cells adapted to suspension culture).

In some embodiments, the cell culture has a volume of about 200 liters. In some embodiments, the cell culture has a volume of about 300 liters. In some embodiments, the cell culture has a volume of about 400 liters. In some embodiments, the cell culture has a volume of about 500 liters. In some embodiments, the cell culture has a volume of about 750 liters. In some embodiments, the cell culture has a volume of about 1,000 liters. In some embodiments, the cell culture has a volume of about 2,000 liters. In some embodiments, the cell culture has a volume of about 3,000 liters. In some embodiments, the cell culture has a volume of about 5,000 liters. In some embodiments, the culture comprises HEK293 cells (eg, HEK293 cells adapted to suspension culture).

In some embodiments, the methods described herein involve adding about 20 liter volumes (e.g., about 21 liters) of two separately produced polynucleotide:transfection reagent complexes to about 400 liters of cells. including transfer to culture. In some embodiments, the methods described herein include transferring three separately generated volumes of about 20 liters of polynucleotide:transfection reagent complexes to about 600 liters of cell culture. . In some embodiments, the methods described herein include transferring four separately generated volumes of about 20 liters of polynucleotide:transfection reagent complexes to about 800 liters of cell culture. . In some embodiments, the methods described herein involve transferring five separately generated volumes of about 20 liters of polynucleotide:transfection reagent complexes to about 1,000 liters of cell culture. including. In some embodiments, the methods described herein include transferring six separately generated volumes of about 20 liters of polynucleotide:transfection reagent complexes to about 1,200 liters of cell culture. including. In some embodiments, the methods described herein involve transferring seven separately generated volumes of about 20 liters of polynucleotide:transfection reagent complexes to about 1,400 liters of cell culture. including. In some embodiments, the methods described herein involve transferring eight separately generated polynucleotide:transfection reagent complexes in a volume of about 20 liters to a cell culture of about 1,600 liters. including. In some embodiments, the methods described herein involve transferring nine separately generated polynucleotide:transfection reagent complexes in a volume of about 20 liters to a cell culture of about 1,800 liters. including. In some embodiments, the methods described herein involve transferring ten separately generated polynucleotide:transfection reagent complexes in a volume of about 20 liters to a cell culture of about 2,000 liters. including. For reference, non-limiting examples of volumes of transfection complex mixtures produced separately are shown in Table 1.

In some embodiments, the methods described herein include transferring one about 40 liter volume (e.g., about 42 liters) of polynucleotide:transfection reagent complex to about 400 liters of cell culture. including. In some embodiments, the methods described herein include transferring two separately generated volumes of about 40 liters of polynucleotide:transfection reagent complexes to about 800 liters of cell culture. . In some embodiments, the methods described herein include transferring four separately generated volumes of about 40 liters of polynucleotide:transfection reagent complexes to about 1,600 liters of cell culture. including. For reference, non-limiting examples of volumes of transfection complex mixtures produced separately are shown in Table 1.

In some embodiments, the rate of transfer is such that mixing, incubating, and transferring takes less than about 90 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, less than about 35 minutes, about Set to complete in less than 30 minutes, less than about 25 minutes, or less than about 20 minutes. In some embodiments, mixing, incubating, and transferring is completed in less than about 30 minutes. In some embodiments, the one or more polynucleotides include genes necessary for production of recombinant AAV particles. In some embodiments, the transfection reagent comprises PEI. In some embodiments, the one or more polynucleotides include one or more helper genes, rep genes, cap genes, and transgenes (eg, genes of interest or rAAV genomes to be packaged). In some embodiments, the culture comprises HEK293 cells (eg, HEK293 cells adapted to suspension culture).

In some embodiments, the methods described herein include transferring two separately generated volumes of about 30 liters of polynucleotide:transfection reagent complexes to about 600 liters of cell culture. . In some embodiments, the methods described herein include transferring three separately generated volumes of about 30 liters of polynucleotide:transfection reagent complexes to about 900 liters of cell culture. . In some embodiments, the methods described herein include transferring four separately generated volumes of about 30 liters of polynucleotide:transfection reagent complexes to about 1,200 liters of cell culture. including. In some embodiments, the methods described herein include transferring five separately generated volumes of about 30 liters of polynucleotide:transfection reagent complexes to about 1,500 liters of cell culture. including. In some embodiments, the methods described herein include transferring six separately generated volumes of about 30 liters of polynucleotide:transfection reagent complexes to about 1,800 liters of cell culture. including. In some embodiments, the methods described herein include transferring about 30 liter volumes of seven separately generated polynucleotide:transfection reagent complexes to about 2,100 liters of cell culture. including. In some embodiments, the methods described herein involve transferring eight separately generated polynucleotide:transfection reagent complexes in a volume of about 30 liters to a cell culture of about 2,400 liters. including. In some embodiments, the methods described herein involve transferring nine separately generated polynucleotide:transfection reagent complexes in a volume of about 30 liters to a cell culture of about 2,700 liters. including. In some embodiments, the methods described herein involve transferring 10 separately generated polynucleotide:transfection reagent complexes in a volume of about 30 liters to a cell culture of about 3,000 liters. including. In some embodiments, the rate of transfer is such that mixing, incubating, and transferring takes less than about 90 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, less than about 35 minutes, about Set to complete in less than 30 minutes, less than about 25 minutes, or less than about 20 minutes. In some embodiments, mixing, incubating, and transferring is completed in less than about 30 minutes. In some embodiments, the one or more polynucleotides include genes necessary for production of recombinant AAV particles. In some embodiments, the transfection reagent comprises PEI. In some embodiments, the culture comprises HEK293 cells (eg, HEK293 cells adapted to suspension culture).

In some embodiments, the mixing, incubating, and transferring takes less than about 90 minutes, less than about 60 minutes, less than about 50 minutes, less than about 40 minutes, less than about 35 minutes, less than about 30 minutes, Complete in less than about 25 minutes, or less than about 20 minutes. In some embodiments, mixing, incubating, and transferring is completed in less than about 60 minutes. In some embodiments, mixing, incubating, and transferring is completed in less than about 50 minutes. In some embodiments, mixing, incubating, and transferring is completed in less than about 40 minutes. In some embodiments, mixing, incubating, and transferring is completed in less than about 35 minutes. In some embodiments, mixing, incubating, and transferring is completed in less than about 30 minutes. In some embodiments, mixing, incubating, and transferring is completed in less than about 25 minutes. In some embodiments, mixing, incubating, and transferring is completed in less than about 20 minutes.

Transferring separately generated polynucleotide:transfection reagent complexes to cell culture can be performed simultaneously or sequentially. Simultaneous transfer includes duplicate transfer. In some embodiments, separately generated polynucleotide:transfection reagent complexes are transferred to cell culture at the same time. In some embodiments, separately generated polynucleotide:transfection reagent complexes are sequentially transferred to cell culture. In some embodiments, transferring the volume of separately generated polynucleotide:transfection reagent complex is initiated prior to completing the transfer of the preceding separately generated volume. In some embodiments, transferring the volume of separately generated polynucleotide:transfection reagent complex is initiated immediately after completing the transfer of the preceding separately generated volume. In some embodiments, transferring the volume of the separately generated polynucleotide:transfection reagent complex occurs within about 5 minutes to about 60 minutes after completing transferring the preceding separately generated volume. will be started in between. In some embodiments, transferring the volume of separately generated polynucleotide:transfection reagent complex is initiated within about 5 minutes of completing the transfer of the preceding separately generated volume. . In some embodiments, transferring the volume of separately generated polynucleotide:transfection reagent complex is initiated within about 10 minutes of completing the transfer of the preceding separately generated volume. . In some embodiments, transferring the volume of separately generated polynucleotide:transfection reagent complex is initiated within about 15 minutes of completing the transfer of the preceding separately generated volume. . In some embodiments, transferring the volume of separately generated polynucleotide:transfection reagent complex is initiated within about 20 minutes of completing the transfer of the preceding separately generated volume. . In some embodiments, transferring the volume of separately generated polynucleotide:transfection reagent complex is initiated within about 30 minutes of completing the transfer of the preceding separately generated volume. . In some embodiments, transferring the volume of separately generated polynucleotide:transfection reagent complex is initiated within about 45 minutes of completing the transfer of the preceding separately generated volume. . In some embodiments, transferring the volume of separately generated polynucleotide:transfection reagent complex is initiated within about 60 minutes of completing the transfer of the preceding separately generated volume. . In some embodiments, the one or more polynucleotides include genes necessary for production of recombinant AAV particles. In some embodiments, the transfection reagent comprises PEI. In some embodiments, the culture comprises HEK293 cells (eg, HEK293 cells adapted to suspension culture).

In some embodiments, separately generated volumes of polynucleotide:transfection reagent complexes are transferred to cell culture over a period of between about 1 hour and about 12 hours. In some embodiments, separately generated volumes of polynucleotide:transfection reagent complexes are transferred to cell culture over a period of between about 1 hour and about 8 hours. In some embodiments, separately generated volumes of polynucleotide:transfection reagent complexes are transferred to cell culture over a period of between about 1 hour and about 6 hours. In some embodiments, separately generated volumes of polynucleotide:transfection reagent complexes are transferred to cell culture over a period of between about 1 hour and about 5 hours. In some embodiments, separately generated volumes of polynucleotide:transfection reagent complexes are transferred to cell culture over a period of between about 1 hour and about 4 hours. In some embodiments, separately generated volumes of polynucleotide:transfection reagent complexes are transferred to cell culture over a period of between about 1 hour and about 3 hours. In some embodiments, separately generated volumes of polynucleotide:transfection reagent complexes are transferred to cell culture over a period of about 12 hours or less. In some embodiments, separately generated volumes of polynucleotide:transfection reagent complexes are transferred to cell culture over a period of about 9 hours or less. In some embodiments, separately generated volumes of polynucleotide:transfection reagent complexes are transferred to cell culture over a period of about 8 hours or less. In some embodiments, separately generated volumes of polynucleotide:transfection reagent complexes are transferred to cell culture over a period of about 7 hours or less. In some embodiments, separately generated volumes of polynucleotide:transfection reagent complexes are transferred to cell culture over a period of about 6 hours or less. In some embodiments, separately generated volumes of polynucleotide:transfection reagent complexes are transferred to cell culture over a period of about 5 hours or less. In some embodiments, separately generated volumes of polynucleotide:transfection reagent complexes are transferred to cell culture over a period of about 5 hours or less. In some embodiments, separately generated volumes of polynucleotide:transfection reagent complexes are transferred to cell culture over a period of about 3 hours or less. In some embodiments, separately generated volumes of polynucleotide:transfection reagent complexes are transferred to cell culture over a period of about 2 hours or less. In some embodiments, the one or more polynucleotides encode genetic information (including genes) necessary for the production of recombinant AAV particles. In some embodiments, the one or more polynucleotides include one or more helper genes, rep genes, cap genes, and transgenes (eg, genes of interest or rAAV genomes to be packaged). In some embodiments, the transfection reagent comprises PEI. In some embodiments, the culture comprises HEK293 cells (eg, HEK293 cells adapted to suspension culture).

In some embodiments, the cell culture contains between about 2 x 10E+6 to about 10E+7 viable cells/ml. In some embodiments, the cell culture contains between about 3 x 10E+6 and about 8 x 10E+6 viable cells/ml. In some embodiments, the cell culture contains about 3 x 10E+6 viable cells/ml. In some embodiments, the cell culture contains about 4 x 10E+6 viable cells/ml. In some embodiments, the cell culture contains about 5 x 10E+6 viable cells/ml. In some embodiments, the cell culture contains about 6 x 10E+6 viable cells/ml. In some embodiments, the cell culture contains about 7 x 10E+6 viable cells/ml. In some embodiments, the cell culture contains about 8 x 10E+6 viable cells/ml. In some embodiments, the culture comprises HEK293 cells (eg, HEK293 cells adapted to suspension culture).

In some embodiments, the population of cells comprises a population of mammalian or insect cells. In some embodiments, the population of cells comprises a population of mammalian cells. In some embodiments, the population of cells comprises a population of HEK293 cells, HEK-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, and/or PerC6 cells. In some embodiments, the population of cells comprises a population of HEK293 cells.

In some embodiments, the cell culture is maintained for about 2 days to about 10 days after transferring the polynucleotide:transfection reagent complex. In some embodiments, the cell culture is maintained for about 3 to about 5 days after transferring the polynucleotide:transfection reagent complex. In some embodiments, the cell culture is maintained for about 5 days to about 14 days or more after transferring the polynucleotide:transfection reagent complex. In some embodiments, the cell culture is maintained for about 2 days to about 7 days after transferring the polynucleotide:transfection reagent complex. In some embodiments, the cell culture is maintained for about 3 to about 5 days after transferring the polynucleotide:transfection reagent complex. In some embodiments, the cell culture is maintained for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after transferring the polynucleotide:transfection reagent complex. Ru. In some embodiments, the cell culture is maintained for at least about 3 days after transferring the polynucleotide:transfection reagent complex. In some embodiments, the cell culture is maintained for about 5 days after transferring the polynucleotide:transfection reagent complex. In some embodiments, the cell culture is maintained for about 6 days after transferring the polynucleotide:transfection reagent complex. In some embodiments, the cell culture is maintained under conditions that allow production of rAAV particles for continued collection. In some embodiments, the culture comprises HEK293 cells (eg, HEK293 cells adapted to suspension culture).

In some embodiments, the methods disclosed herein reduce the amount of recombinant virus compared to reference methods that involve a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complexes. Increase the production of particles (eg, rAAV particles). In some embodiments, the methods disclosed herein increase production of recombinant virus by at least about 50%, at least about 75%, or at least about 100%. In some embodiments, the methods disclosed herein increase the production of recombinant virus by at least about 2-fold, at least about 3-fold, or at least about 5-fold. In some embodiments, the methods disclosed herein increase production of rAAV by at least about 2-fold. In some embodiments, increased production is quantified by comparing recombinant virus (eg, rAAV) titers of the production cultures. In some embodiments, recombinant virus (eg, rAAV) titer is measured as genome copies (GC) per milliliter of production culture. In some embodiments, the recombinant virus is rAAV. In some embodiments, the rAAV particle comprises a capsid protein from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9. In some embodiments, the rAAV particles are AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. PHB, and AAV. It has a capsid serotype selected from the group consisting of 7m8. In some embodiments, the rAAV particle comprises a capsid protein with high sequence homology to AAV8 or AAV9, e.g., AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, and AAV. It has hu37.

In some embodiments, the methods disclosed herein increase the production of rAAV particles while maintaining or improving the quality attributes of the rAAV particles and compositions containing them. In some embodiments, the quality of rAAV particles and compositions containing them is determined by the concentration of rAAV particles (e.g., GC/ml), the percentage of particles that contain copies of the rAAV genome, the proportion of particles that do not contain a genome, the rAAV particle infectivity, rAAV particle stability, residual host cell protein concentration or residual host cell nucleic acid concentration (e.g., host cell genomic DNA, plasmids encoding rep and cap genes, plasmids encoding helper functions, plasmid encoding the rAAV genome). In some embodiments, the quality of rAAV particles produced by the methods disclosed herein, or compositions comprising the same, is determined by mixing, incubating, and transferring the same volumes of polynucleotide:transfection reagent complexes. The quality is the same as that of rAAV particles or compositions produced by reference methods involving a single step. In some embodiments, the quality of rAAV particles produced by the methods disclosed herein, or compositions comprising the same, is determined by mixing, incubating, and transferring the same volumes of polynucleotide:transfection reagent complexes. Better than the quality of rAAV particles or compositions produced by reference methods involving a single step.

In some embodiments, the methods disclosed herein produce between about 1×10e+10 GC/ml and about 1×10e+13 GC/ml of rAAV particles. In some embodiments, the methods disclosed herein produce between about 1×10e+10 GC/ml and about 1×10e+11 GC/ml of rAAV particles. In some embodiments, the methods disclosed herein produce between about 5×10e+10 GC/ml and about 1×10e+12 GC/ml of rAAV particles. In some embodiments, the methods disclosed herein produce between about 5×10e+10 GC/ml and about 1×10e+13 GC/ml of rAAV particles. In some embodiments, the methods disclosed herein produce between about 1×10e+11 GC/ml and about 1×10e+13 GC/ml of rAAV particles. In some embodiments, the methods disclosed herein produce between about 5×10e+10 GC/ml and about 5×10e+12 GC/ml of rAAV particles. In some embodiments, the methods disclosed herein produce between about 1×10e+11 GC/ml and about 5×10e+12 GC/ml of rAAV particles. In some embodiments, the methods disclosed herein produce greater than about 1×10e+11 GC/ml of rAAV particles. In some embodiments, the methods disclosed herein produce greater than about 5×10e+11 GC/ml of rAAV particles. In some embodiments, the methods disclosed herein produce greater than about 1×10e+12 GC/ml of rAAV particles. In some embodiments, the rAAV particle comprises a capsid protein from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9. In some embodiments, the rAAV particles are AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. PHB, and AAV. 7m8. In some embodiments, the rAAV particle comprises a capsid protein with high sequence homology to AAV8 or AAV9, e.g., AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, and AAV. Contains hu37.

In some embodiments, the methods disclosed herein produce at least about 5×10e+10 GC/ml rAAV particles. In some embodiments, the methods disclosed herein produce at least about 1×10e+11 GC/ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 5×10e+11 GC/ml of rAAV particles. In some embodiments, the methods disclosed herein produce at least about 1×10e+12 GC/ml rAAV particles. In some embodiments, the methods disclosed herein produce at least about 5×10e+12 GC/ml rAAV particles. In some embodiments, the methods disclosed herein produce at least about 1×10e+13 GC/ml rAAV particles. In some embodiments, the methods disclosed herein produce at least about 5×10e+13 GC/ml rAAV particles. In some embodiments, the rAAV particle comprises a capsid protein from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9. In some embodiments, the rAAV particles are AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. PHB, and AAV. 7m8. In some embodiments, the rAAV particle comprises a capsid protein with high sequence homology to AAV8 or AAV9, e.g., AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, and AAV. Contains hu37.

Numerous cell culture-based systems for the production of rAAV particles are known in the art, any of which can be used in practicing the methods disclosed herein. The rAAV production culture for producing rAAV virions comprises (1) a suitable host cell (e.g., a human-derived cell line (e.g., HeLa, A549, or HEK293 cells and derivatives thereof (HEK293T cells, HEK293F cells)); (2) wild-type or mutant adenoviruses (e.g., temperature-sensitive adenoviruses), herpesviruses, baculoviruses, or provide helper functions; (3) AAV rep and cap genes and gene products; (4) transgenes (e.g., therapeutic transgenes) flanking AAV ITR sequences; and (5) A suitable medium and medium components are required to support rAAV production.

Those skilled in the art will recognize the AAV rep and cap genes, the AAV helper genes (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and the rAAV genome (1 flanking the inverted terminal repeats (ITRs)). We are aware of numerous ways in which rAAV can be produced or packaged by introducing one or more genes of interest into cells. The expression "adenovirus helper functions" refers to multiple viral helper genes that are expressed within the cell (as RNA or protein) to allow AAV to grow efficiently within the cell. Those skilled in the art will appreciate that helper viruses, including adenovirus and herpes simplex virus (HSV), promote AAV replication and that certain genes have been identified that provide essential functions; It is understood that changes to the cellular environment that promote AAV gene expression and replication can be induced. In some embodiments of the methods disclosed herein, the AAV rep and cap genes, helper genes, and rAAV genome are isolated from one or more plasmids encoding the AAV rep and cap genes, helper genes, and rAAV genome. The vector is introduced into cells by transfection.

Molecular biology techniques for developing plasmid or viral vectors encoding AAV rep and cap genes, helper genes, and/or rAAV genomes are generally known in the art. In some embodiments, the AAV rep and cap genes are encoded by one plasmid vector. In some embodiments, the AAV helper genes (eg, adenovirus E1a, E1b, E4, E2a, and VA genes) are encoded by one plasmid vector. In some embodiments, the E1a or E1b gene is stably expressed by the host cell and the remaining AAV helper genes are introduced into the cell by transfection with one viral vector. In some embodiments, the E1a and E1b genes are stably expressed by the host cell, and the E4, E2a, and VA genes are introduced into the cell by transfection with one plasmid vector. In some embodiments, the one or more helper genes are stably expressed by the host cell, and the one or more helper genes are introduced into the cell by transfection with one plasmid vector. In some embodiments, the helper gene is stably expressed by the host cell. In some embodiments, the AAV rep and cap genes are encoded by one viral vector. In some embodiments, the AAV helper genes (eg, adenovirus E1a, E1b, E4, E2a, and VA genes) are encoded by one viral vector. In some embodiments, the E1a or E1b gene is stably expressed by the host cell and the remaining AAV helper genes are introduced into the cell by transfection with one viral vector. In some embodiments, the E1a and E1b genes are stably expressed by the host cell, and the E4, E2a, and VA genes are introduced into the cell by transfection with one viral vector. In some embodiments, the one or more helper genes are stably expressed by the host cell, and the one or more helper genes are introduced into the cell by transfection with one viral vector. In some embodiments, the AAV rep and cap genes, adenoviral helper functions necessary for packaging, and the rAAV genome to be packaged are added to cells by transfection with one or more polynucleotides, e.g., a vector. will be introduced in In some embodiments, the methods disclosed herein include a mixture of three polynucleotides, one encoding the cap and rep genes and one containing the adenovirus helper functions necessary for packaging. For example, transfecting a cell with an adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene), one encoding the rAAV genome to be packaged. In some embodiments, the AAV cap gene is an AAV8 or AAV9 cap gene. In some embodiments, the AAV cap gene is AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. PHB, or AAV. 7m8 cap gene. In some embodiments, the AAV cap gene is a capsid protein with high sequence homology to AAV8 or AAV9, e.g., AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, and AAV. Code hu37. In some embodiments, the vector encoding the rAAV genome to be packaged includes the gene of interest flanked by AAV ITRs. In some embodiments, the AAV ITR is AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15 or AAV. HSC16, or from other AAV serotypes.

Any combination of vectors can be used to introduce AAV rep and cap genes, AAV helper genes, and the rAAV genome into cells in which rAAV particles are produced or packaged. In some embodiments of the methods disclosed herein, a first plasmid vector encoding an rAAV genome comprising a gene of interest flanking an AAV inverted terminal repeat (ITR) and encoding AAV rep and cap genes; A second vector encoding a helper gene and a third vector encoding a helper gene can be used. In some embodiments, a mixture of three vectors can be co-transfected into cells.

In some embodiments, a combination of transfection and infection is used by using viral vectors in addition to plasmid vectors.

In some embodiments, one or more of the rep and cap genes and the AAV helper gene are constitutively expressed by the cell and there is no need to transfect or transduce the cell. In some embodiments, the cell constitutively expresses the rep and/or cap genes. In some embodiments, the cell constitutively expresses one or more AAV helper genes. In some embodiments, the cell constitutively expresses E1a. In some embodiments, the cell contains a stable transgene encoding an rAAV genome.

In some embodiments, the AAV rep, cap, and helper genes (eg, Ela, E1b, E4, E2a, or VA genes) can be of any AAV serotype. Similarly, an AAV ITR can be any AAV serotype. For example, in some embodiments, the AAV ITRs include AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15 or AAV. HSC16, or from other AAV serotypes (eg, hybrid serotypes having sequences from two or more serotypes). In some embodiments, the AAV cap gene is from the AAV9 or AAV8 cap gene. In some embodiments, the AAV cap gene is AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15 or AAV. HSC16, or from other AAV serotypes (eg, hybrid serotypes having sequences from two or more serotypes). In some embodiments, the AAV rep and cap genes for rAAV particle production are from different serotypes. For example, the rep gene is from AAV2, while the cap gene is from AAV9.

Any suitable medium known in the art can be used to produce recombinant virus particles (eg, rAAV particles) according to the methods disclosed herein. Such media include, but are not limited to, modified Eagle's medium (MEM), Dulbecco's modified Eagle's medium (DMEM), and U.S. Pat. media produced by Hyclone Laboratories and JRH, including Sf-900 II SFM media, as described in J.D. In some embodiments, the medium comprises Dynamis™ medium, FreeStyle™ 293 expression medium, or Expi293™ expression medium from Invitrogen/ThermoFisher. In some embodiments, the medium comprises Dynamis™ medium. In some embodiments, the methods disclosed herein use cell cultures that include serum-free media, animal component-free media, or chemically defined media. In some embodiments, the medium is an animal component free medium. In some embodiments, the medium includes serum. In some embodiments, the medium comprises fetal bovine serum. In some embodiments, the medium is a glutamine-free medium. In some embodiments, the medium includes glutamine. In some embodiments, the medium is supplemented with one or more of nutrients, salts, buffers, and additives (eg, antifoams). In some embodiments, the medium is supplemented with glutamine. In some embodiments, the medium is supplemented with serum. In some embodiments, the medium is supplemented with fetal bovine serum. In some embodiments, the medium is supplemented with a poloxamer, such as Kolliphor® P 188 Bio. In some embodiments, the medium is a basal medium. In some embodiments, the medium is a feed medium.

Recombinant virus (e.g., rAAV) production cultures are routinely grown under various conditions (over a wide temperature range, for various lengths of time, etc.) appropriate to the particular host cell being utilized. be able to. As is known in the art, rAAV virus production cultures include suspension-adapted host cells, such as HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK -1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per. Includes C6 cells, chicken embryo cells, and SF-9 cells, which can be cultured in a variety of ways, including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as Wave bag systems. Numerous suspension cultures for producing rAAV particles are known in the art, including, for example, U.S. Pat. No. 20120122155, each of which is incorporated by reference in its entirety. In some embodiments, the recombinant virus is recombinant AAV.

Any cell or cell line known in the art to produce recombinant viral particles (e.g., rAAV particles) can be used in any one of the methods disclosed herein. . In some embodiments, the methods of producing recombinant virus particles (e.g., rAAV particles) or increasing the production of recombinant virus particles (e.g., rAAV particles) disclosed herein are directed to HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, LLC-MK cells, MDCK cells, RAF cells, RK cells, TCMK- 1 cell, PK15 cell, BHK cell, BHK-21 cell, NS-1 cell, BHK cell, 293 cell, RK cell, Per. Use C6 cells, chicken embryo cells, or SF-9 cells. In some embodiments, the methods disclosed herein use mammalian cells. In some embodiments, the methods disclosed herein use insect cells, such as SF-9 cells. In some embodiments, the methods disclosed herein use cells adapted for growth in suspension culture. In some embodiments, the methods disclosed herein use HEK293 cells adapted for growth in suspension culture. In some embodiments, the recombinant viral particles are recombinant AAV particles.

In some embodiments, the cell cultures disclosed herein are suspension cultures. In some embodiments, the large scale suspension cell culture disclosed herein comprises HEK293 cells adapted for growth in suspension culture. In some embodiments, the cell culture disclosed herein comprises a serum-free medium, an animal component-free medium, or a chemically defined medium. In some embodiments, the cell cultures disclosed herein include serum-free media. In some embodiments, suspension-adapted cells are cultured in shake flasks, spinner flasks, cell bags, or bioreactors.

In some embodiments, the cell culture disclosed herein comprises a serum-free medium, an animal component-free medium, or a chemically defined medium. In some embodiments, the cell cultures disclosed herein include serum-free media.

In some embodiments, the cell cultures disclosed herein include an anti-aggregation agent. In some embodiments, the cell culture disclosed herein comprises dextran sulfate. In some embodiments, a cell culture disclosed herein comprises between about 0.1 mg/L and about 10 mg/L dextran sulfate. Methods for transfecting host cells in a culture medium containing dextran sulfate are described in U.S. Provisional Application No. 63/139,992, filed January 21, 2021, entitled "Improved production of recombinant polypeptides and viruses." ) (incorporated herein by reference in its entirety).

In some embodiments, the large scale suspension cell cultures disclosed herein include high density cell cultures. In some embodiments, the culture has a total cell density between about 1 x 10E+06 cells/ml and about 30 x 10E+06 cells/ml. In some embodiments, greater than about 50% of the cells are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293-derived cells (eg, HEK293T cells, HEK293F cells), Vero cells, or SF-9 cells. In further embodiments, the cells are HEK293 cells.

The methods disclosed herein can be used in the production of rAAV particles comprising capsid proteins from any AAV capsid serotype. In some embodiments, the rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, and AAV. Contains capsid proteins from AAV capsid serotypes selected from HSC16. In some embodiments, the rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, or AAV. Includes capsid proteins that are derivatives, modifications, or pseudotypes of HSC16 capsid protein.

In some embodiments, the rAAV particle comprises a capsid protein from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9.

In some embodiments, the rAAV particles are AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. PHB, and AAV. 7m8. In some embodiments, the rAAV particle comprises a capsid protein with high sequence homology to AAV8 or AAV9, e.g., AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, and AAV. Contains hu37.

In some embodiments, the rAAV particle comprises a capsid protein that is a derivative, modification, or pseudotype of AAV8 capsid protein or AAV9 capsid protein. In some embodiments, the rAAV particles are at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, to the VP1, VP2, and/or VP3 sequences of the AAV8 capsid protein. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical to AAV8 capsid protein. including.

In some embodiments, the rAAV particle comprises a capsid protein that is a derivative, modification, or pseudotype of the AAV9 capsid protein. In some embodiments, the rAAV particles are at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, to the VP1, VP2, and/or VP3 sequences of the AAV9 capsid protein. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical to AAV9 capsid protein. including.

In some embodiments, the rAAV particles are AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. PHB, or AAV. At least 80% identity to the VP1, VP2, and/or VP3 sequences of the 7m8 capsid protein, such as 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., ie, up to 100% identity. In some embodiments, the rAAV particles are AAV capsid proteins with high sequence homology to AAV8 or AAV9, e.g., AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, and AAV. At least 80% identity to the VP1, VP2, and/or VP3 sequences of hu37, such as 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, Includes capsid proteins with 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., ie, up to 100% identity.

In additional embodiments, the rAAV particles include mosaic capsids. In additional embodiments, the rAAV particles include pseudotyped rAAV particles. In additional embodiments, the rAAV particles include capsids that include capsid protein chimeras of two or more AAV capsid serotypes.

rAAV Particles The methods provided are suitable for use in the production of any isolated recombinant AAV particles. As such, rAAV may include any serotype, modification, or derivative known in the art, or any combination thereof (e.g., including two or more serotypes (e.g., rAAV2, rAAV8, and a population of rAAV particles) comprising two or more of the rAAV9 particles). In some embodiments, the rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15 or AAV. HSC16, or other rAAV particles, or a combination of two or more of these.

In some embodiments, the rAAV particles are AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15 or AAV. having a capsid protein from an AAV serotype selected from HSC16, or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles are, for example, AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, rAAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, or AAV. At least 80% identical to the VP1, VP2, and/or VP3 sequence of an AAV capsid serotype selected from HSC16, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., ie, up to 100% identical.

In some embodiments, the rAAV particles are AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15 or AAV. A capsid protein from an AAV capsid serotype selected from HSC16, or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles are, for example, AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, or AAV. At least 80% identical to the VP1, VP2, and/or VP3 sequence of an AAV capsid serotype selected from HSC16, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., ie, up to 100% identical.

In some embodiments, the rAAV particles are as described by Zinn et al. , 2015, Cell Rep. 12(6):1056-1068 (incorporated by reference in its entirety). In certain embodiments, the rAAV particles are described in U.S. Pat. (incorporated herein by reference in its entirety), including capsids with one of the following amino acid insertions: LGETTRP or LALGETTRP. In some embodiments, the rAAV particles are described in U.S. Pat. each of which is incorporated herein by reference in its entirety). Contains 7m8 capsids. In some embodiments, the rAAV particles are any AAV capsid disclosed in U.S. Patent No. 9,585,971, such as AAVPHP. Contains B. In some embodiments, the rAAV particles are any of the particles disclosed in U.S. Patent No. 9,840,719 and WO2015/013313, each of which is incorporated herein by reference in its entirety. AAV capsids, such as AAV. Contains Rh74 and RHM4-1. In some embodiments, the rAAV particle is any AAV capsid disclosed in WO2014/172669 (herein incorporated by reference in its entirety), such as AAV rh. 74 included. In some embodiments, rAAV particles are as described by Georgiadis et al. , 2016, Gene Therapy 23:857-862 and Georgiadis et al. , 2018, Gene Therapy 25:450, each of which is incorporated by reference in its entirety. In some embodiments, the rAAV particle comprises any AAV capsid disclosed in WO2017/070491, herein incorporated by reference in its entirety, such as AAV2tYF. In some embodiments, the rAAV particles are as described by Puzzo et al. , 2017, Sci. Transl. Med. 29(9):418 (incorporated by reference in its entirety). In some embodiments, the rAAV particle is any AAV capsid disclosed in US Pat. No. 8,628,966, US Pat. No. 8,927,514, US Pat. , each of which is incorporated herein by reference in its entirety. ).

In some embodiments, rAAV particles are disclosed in the following patents and patent applications, each of which is incorporated herein by reference in its entirety: U.S. Patent No. 7,282,199; No. 906,111, No. 8,524,446, No. 8,999,678, No. 8,628,966, No. 8,927,514, No. 8,734,809, No. US 9,284, No. 357, No. 9,409,953, No. 9,169,299, No. 9,193,956, No. 9458517, and No. 9,587,282, U.S. Patent Application Publication No. 2015/0374803, No. Any of 2015/0126588, 2017/0067908, 2013/0224836, 2016/0215024, 2017/0051257, and International Patent Application No. PCT/US2015/034799, PCT/EP2015/053335 including the AAV capsid disclosed in . In some embodiments, the rAAV particles are AAV capsid VP1, VP2 as disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: , and/or at least 80% identical to the VP3 sequence, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical: U.S. Pat. No. 8,524,446, No. 8,999,678, No. 8,628,966, No. 8,927,514, No. 8,734,809, No. 9,284,357, No. 9, No. 409,953, No. 9,169,299, No. 9,193,956, No. 9458517, and No. 9,587,282; No. 2017/0067908, No. 2013/0224836, No. 2016/0215024, No. 2017/0051257, and International Patent Application No. PCT/US2015/034799, No. PCT/EP2015/053335.

In some embodiments, the rAAV particle comprises International Patent Application Publication No. WO 2003/052051 (see, e.g., SEQ ID NO: 2), WO 2005/033321 (see, e.g., SEQ ID NO: 123 and 88), WO 03/ 042397 (see, for example, SEQ ID NOs: 2, 81, 85, and 97), WO2006/068888 (see, for example, SEQ ID NOs: 1 and 3-6), WO2006/110689 (for example, see SEQ ID NOs: 5-6), No. 38), WO 2009/104964 (see e.g. SEQ ID Nos. 1-5, 7, 9, 20, 22, 24 and 31), WO 2010/127097 (see e.g. SEQ ID Nos. 5-38) ), as well as WO 2015/191508 (see, e.g., SEQ ID NOs: 80-294), and US Patent Application Publication No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10), the contents of each of which are incorporated herein by reference. (incorporated herein in its entirety). In some embodiments, the rAAV particles are at least 80% identical, e.g., 85%, 85%, 87%, 88%, to the AAV capsid VP1, VP2 and/or VP3 sequences disclosed below. , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical. has: International Patent Application Publication No. WO 2003/052051 (see, e.g., SEQ ID NO: 2), WO 2005/033321 (see, e.g., SEQ ID NO: 123 and 88), WO 03/042397 (see, e.g., SEQ ID NO: 2). . 104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24, and 31), WO2010/127097 (see, e.g., SEQ ID NOs: 5-38), and WO2015/191508. (see, eg, SEQ ID NO: 80-294), as well as US Patent Application Publication No. 20150023924 (see, eg, SEQ ID NO: 1, 5-10).

Nucleic acid sequences of AAV-based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in: U.S. Pat. , 524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, US 9,284,357, 9,409 , 953, 9,169,299, 9,193,956, 9458517, and 9,587,282, U.S. Patent Application Publication No. 2015/0374803, 2015/0126588, 2017/0067908, 2013/0224836, 2016/0215024, 2017/0051257, International Patent Application No. PCT/US2015/034799, PCT/EP2015/053335, WO2003/052051, WO20 05 / 033321, Wo03 / 042397, WO2006 / 068888, WO2006 / 110689, WO2009 / 104964, WO2010 / 127097, and No. Published US Patent Publishing 20150023924.

The methods provided are suitable for use in the generation of recombinant AAV encoding transgenes. In certain embodiments, the transgene is selected from Tables 2A-2C. In some embodiments, the rAAV genome comprises the following components: (1) AAV inverted terminal repeats flanking the expression cassette; and (2) regulatory control elements, e.g., a) promoter/enhancer, b) polyAAV a signal; and c) optionally an intron; and (3) a nucleic acid sequence encoding a transgene. In other embodiments for expressing intact or substantially intact monoclonal antibodies (mAbs), the rAAV genome comprises the following components: (1) an AAV inverted terminal repeat flanking the expression cassette; ) regulatory control elements, such as a) a promoter/enhancer, b) a polyA signal, and c) optionally an intron; (3) a light chain Fab and a heavy chain Fab of the antibody, or at least a heavy or light chain Fab; and, optionally, a nucleic acid sequence encoding a heavy chain Fc region. In yet other embodiments for expressing intact or substantially intact mAbs, the rAAV genome comprises a vector comprising the following components: (1) AAV inverted terminal repeats flanking the expression cassette; (2) ) regulatory control elements, such as a) a promoter/enhancer, b) a polyA signal, and c) optionally an intron; and (3) a nucleic acid sequence encoding a heavy chain Fab of: anti-VEGF (e.g., cebacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 (e.g., ascribacumab), anti-C5 (e.g., tesidolumab and eculizumab), anti-CD105 (e.g., carotuximab), anti-CC1Q (e.g., For example, ANX-007), anti-TNFα (e.g., adalimumab, infliximab, and golimumab), anti-RGMa (e.g., elezanumab), anti-TTR (e.g., NI-301 and PRX-004), anti-CTGF (e.g., pamrevlumab), anti-IL6R (e.g., satralizumab and sarilumab), anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixekizumab and secukinumab), anti-IL-5 (e.g., mepolizumab), anti-IL12/IL23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-ITGF7 mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-pKal mAb (e.g., lanadelumab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti- BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., densomab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab*), anti-OxPL (e.g. , E06), anti-fD (e.g., lampalizumab), or anti-MMP9 (e.g., andecaliximab); optionally, an Fc polypeptide of the same isotype as the native form of the therapeutic antibody, e.g. or IgG4, or modified Fc thereof; and nucleic acid sequences encoding the light chains of: anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651), anti-ALK1 ( anti-C5 (e.g., tesidolumab and eculizumab), anti-CD105 or anti-ENG (e.g., carotuximab), anti-CC1Q (e.g., ANX-007), anti-TNFα (e.g., adalimumab, infliximab, and golimumab); ), anti-RGMa (e.g., elezanumab), anti-TTR (e.g., NI-301 and PRX-004), anti-CTGF (e.g., pamrevlumumab), anti-IL6R (e.g., satralizumab and sarilumab), anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixekizumab and secukinumab), anti-IL-5 (e.g., mepolizumab), anti-IL12/IL23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-ITGF7 mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-pKal mAbs (e.g., lanadelumab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab). , anti-RANKL (e.g., densomab), anti-PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab), anti-OxPL (e.g., E06), anti-fD (e.g., lampalizumab), or anti-MMP9 (e.g., and caliximab). The heavy chain (Fab and optionally the Fc region) and the light chain are then separated by a self-cleaving furin (F)/F2A or furin (F)/F2A or flexible linker such that the heavy and light chain polypeptides are equal. Ensure expression in quantity.

In some embodiments, provided herein are rAAV viral vectors encoding anti-VEGF Fabs. In specific embodiments, provided herein are rAAV8-based viral vectors encoding anti-VEGF Fabs. In a more specific embodiment, provided herein is an rAAV8-based viral vector encoding ranibizumab. In some embodiments, provided herein is an rAAV viral vector encoding iduronidase (IDUA). In a specific embodiment, provided herein is an rAAV9-based viral vector encoding IDUA. In some embodiments, provided herein is an rAAV viral vector encoding iduronate 2-sulfatase (IDS). In a specific embodiment, provided herein is an rAAV9-based viral vector encoding an IDS. In some embodiments, provided herein are rAAV viral vectors encoding low density lipoprotein receptor (LDLR). In specific embodiments, provided herein are rAAV8-based viral vectors encoding LDLR. In some embodiments, provided herein is an rAAV viral vector encoding tripeptidyl peptidase 1 (TPP1) protein. In a specific embodiment, provided herein is an rAAV9-based viral vector encoding TPP1. In some embodiments, provided herein are rAAV viral vectors encoding non-membrane bound splice variants of VEGF receptor 1 (sFlt-1). In some embodiments, the present invention describes gamma-sarcoglycan, Rab escort protein 1 (REP1/CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide gated channel alpha 3 (CNGA3), cyclic nucleotide gated channel beta 3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinosxin (RS1) ), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamate decarboxylase (GAD), glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 ( AQP1), dystrophin, microdystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) ), arylsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha 1 - Antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neurturin (NRTN), neurotrophin-3 (NT-3/NTF3) , porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrial code NADH: ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dysferlin, MER proto-oncogene tyrosine rAAV viral vectors encoding a cystic fibrosis transmembrane conductance regulator (CFTR), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1) Fc fusion are provided.

In additional embodiments, the rAAV particle comprises a pseudotyped AAV capsid. In some embodiments, the pseudotyped AAV capsid is an rAAV2/8 or rAAV2/9 pseudotyped AAV capsid. Methods for producing and using pseudotyped rAAV particles are known in the art (eg, Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001 ) see ).

In additional embodiments, the rAAV particles include capsids that include capsid proteins that are chimeras of two or more AAV capsid serotypes. In some embodiments, the capsid protein is AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, or AAV. It is a chimera of two or more AAV capsid proteins from AAV serotypes selected from HSC16.

In certain embodiments, single chain AAV (ssAAV) can be used. In certain embodiments, self-complementary vectors, such as scAAV, can be used (e.g., Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16:1248-1254; and U.S. Pat. (incorporated into the book).

In some embodiments, the rAAV particle comprises a capsid protein from an AAV capsid serotype selected from AAV8 or AAV9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9.

In some embodiments, the rAAV particle comprises a capsid protein that is a derivative, modification, or pseudotype of AAV8 capsid protein or AAV9 capsid protein. In some embodiments, the rAAV particles are at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, to the VP1, VP2, and/or VP3 sequences of the AAV8 capsid protein. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical to AAV8 capsid protein. including.

In some embodiments, the rAAV particle comprises a capsid protein that is a derivative, modification, or pseudotype of the AAV9 capsid protein. In some embodiments, the rAAV particles are at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, to the VP1, VP2, and/or VP3 sequences of the AAV9 capsid protein. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical to AAV9 capsid protein. including.

In additional embodiments, the rAAV particles include mosaic capsids. Mosaic AAV particles are composed of a mixture of viral capsid proteins from different serotypes of AAV. In some embodiments, the rAAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, and AAV. It contains a mosaic capsid containing capsid proteins of serotypes selected from HSC16. In some embodiments, the rAAV particles are AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh. 8, AAVrh. 10, AAVhu. 37, AAVrh. 20, and AAVrh. It contains a mosaic capsid containing capsid proteins of serotypes selected from 74.

In additional embodiments, the rAAV particles include pseudotyped rAAV particles. In some embodiments, pseudotyped rAAV particles include (a) a nucleic acid vector comprising an AAV ITR, and (b) an AAVx (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10). , AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV16, AAV.RH10, AAV.RH20, AAV.RH39, AAV.RH39, AAV.RHM4-1, AAV.RHM4-1, AAV.HU37, AAV.AV.AV.ANC80 AAV. Anc80L65 , AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7 , AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16). . In additional embodiments, the rAAV particles are AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh. 8, AAVrh. 10, AAVhu. 37, AAVrh. 20, and AAVrh. The present invention includes pseudotyped rAAV particles composed of capsid proteins of AAV serotypes selected from 74. In additional embodiments, the rAAV particles include pseudotyped rAAV particles that include AAV8 capsid protein. In additional embodiments, the rAAV particle comprises a pseudotyped rAAV particle comprised of AAV9 capsid protein. In some embodiments, the pseudotyped rAAV8 or rAAV9 particles are rAAV2/8 or rAAV2/9 pseudotyped particles. Methods for producing and using pseudotyped rAAV particles are known in the art (eg, Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001 ) see ).

In additional embodiments, the rAAV particles include capsids that include capsid proteins that are chimeras of two or more AAV capsid serotypes. In some embodiments, the rAAV particle comprises AAV8 capsid protein and AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, and AAV. AAV capsid protein chimeras with one or more AAV capsid proteins from AAV serotypes selected from HSC16. In some embodiments, the rAAV particles contain AAV8 capsid protein and AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, rAAVrh10, AAVrh. 8, AAVrh. 10, AAVhu. 37, AAVrh. 20, and AAVrh. AAV capsid proteins that are chimeras with one or more AAV capsid proteins from AAV serotypes selected from 74. In some embodiments, the rAAV particle comprises AAV9 capsid protein and AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, and AAV. An AAV capsid protein that is a chimera with a capsid protein of one or more AAV capsid serotypes selected from HSC16. In some embodiments, the rAAV particles contain AAV9 capsid protein and AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh. 8, AAVrh. 10, AAVhu. 37, AAVrh. 20, and AAVrh. AAV capsid proteins that are chimeras with capsid proteins of one or more AAV capsid serotypes selected from 74.

Methods for Isolating rAAV Particles In some embodiments, the present disclosure provides a method for producing a composition comprising isolated recombinant adeno-associated virus (rAAV) particles containing impurities. A method is provided that includes isolating rAAV particles from a feed (eg, an rAAV production culture). In some embodiments, the methods of manufacturing formulations comprising isolated recombinant adeno-associated virus (rAAV) particles disclosed herein include (a) a feed containing impurities (e.g., an rAAV-producing culture). (b) formulating the isolated rAAV particles to produce a formulation.

In some embodiments, the present disclosure further provides a method for producing a formulation comprising a pharmaceutical unit dose of isolated recombinant adeno-associated virus (rAAV) particles, the method comprising: A method is provided that includes isolating rAAV particles from an rAAV-producing culture) and formulating the isolated rAAV particles.

Isolated rAAV particles can be isolated using methods known in the art. In some embodiments, the method of isolating rAAV particles involves downstream processing, e.g., harvesting of cell cultures, clarification of harvested cell cultures (e.g., by centrifugation or depth filtration), tangential flow filtration. , affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, sterile filtration, or any combination(s) thereof. In some embodiments, downstream processing includes harvesting cell cultures, clarifying harvested cell cultures (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, comprising at least two, at least three, at least four, at least five, or at least six of cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, and sterile filtration. In some embodiments, downstream processing includes harvesting cell cultures, clarifying harvested cell cultures (e.g., by depth filtration), sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. including. In some embodiments, downstream processing includes clarification of the harvested cell culture, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, downstream processing includes clarification of the harvested cell culture by depth filtration, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, clarification of the harvested cell culture includes sterile filtration. In some embodiments, downstream processing does not include centrifugation. In some embodiments, the rAAV particle comprises a capsid protein of serotype AAV8. In some embodiments, the rAAV particle comprises a capsid protein of the AAV9 serotype.

In some embodiments, a method of isolating rAAV particles according to the methods disclosed herein includes harvesting a cell culture, clarification of the harvested cell culture (e.g., by depth filtration), first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography with a quaternary amine ligand), a second tangential flow filtration, and a second tangential flow filtration. 2 sterile filtration. In some embodiments, the methods of isolating rAAV particles disclosed herein include harvesting a cell culture, clarifying the harvested cell culture (e.g., by depth filtration), first sterile filtration. , affinity chromatography, anion exchange chromatography (eg, monolith anion exchange chromatography or AEX chromatography with quaternary amine ligands), tangential flow filtration, and second sterile filtration. In some embodiments, a method of isolating rAAV particles produced according to the methods disclosed herein comprises: clarification of a harvested cell culture, a first sterile filtration, a first tangential flow filtration. , affinity chromatography, anion exchange chromatography (eg, monolith anion exchange chromatography or AEX chromatography with a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. In some embodiments, the methods of isolating rAAV particles disclosed herein include clarification of harvested cell cultures, first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., (monolith anion exchange chromatography or AEX chromatography using grade amine ligands), tangential flow filtration, and a second sterile filtration. In some embodiments, a method of isolating rAAV particles produced according to the methods disclosed herein comprises: clarification of the harvested cell culture by depth filtration, a first sterile filtration, a first including tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography with a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. . In some embodiments, the methods of isolating rAAV particles disclosed herein include clarification of harvested cell cultures by depth filtration, first sterile filtration, affinity chromatography, anion exchange chromatography ( Examples include monolith anion exchange chromatography or AEX chromatography using quaternary amine ligands), tangential flow filtration, and a second sterile filtration. In some embodiments, the method does not include centrifugation. In some embodiments, clarification of the harvested cell culture includes sterile filtration. In some embodiments, the rAAV particle comprises a capsid protein of serotype AAV8. In some embodiments, the rAAV particle comprises a capsid protein of the AAV9 serotype.

The art uses techniques for transfection, stable cell line generation, and generation of rAAV particles, including infectious hybrid virus generation systems (including adenovirus-AAV hybrids, herpesvirus-AAV hybrids, and baculovirus-AAV hybrids). A number of cell culture-based systems are known. Any rAAV production culture for producing rAAV virions may be derived from (1) a suitable host cell, e.g., a human-derived cell line (e.g., HeLa, A549, or HEK293 cells and derivatives thereof (HEK293T cells, HEK293F cells); )), mammalian cell lines (e.g., Vero), or, in the case of baculovirus production systems, insect-derived cell lines (e.g., SF-9); (2) wild-type or mutant adenoviruses (e.g., temperature-sensitive (3) AAV rep and cap genes and gene products; (4) flanking AAV ITR sequences; (5) a suitable medium and medium components to support rAAV production. Any suitable medium known in the art can be used to generate rAAV vectors. Such media include, but are not limited to, modified Eagle's medium (MEM), Dulbecco's modified Eagle's medium (DMEM), and U.S. Pat. media produced by Hyclone Laboratories and JRH, including Sf-900 II SFM media, as described in J.D.

rAAV production cultures can be routinely grown under a variety of conditions (over a wide temperature range, for varying lengths of time, etc.) appropriate to the particular host cell being utilized. As is known in the art, rAAV-producing cultures can be cultured in suitable attachment-dependent containers (e.g., roller bottles, hollow fiber filters, microcarriers, and packed or fluidized bed bioreactors). Contains attachment-dependent cultures capable of rAAV vector-producing cultures can also be produced using suspension-adapted host cells, such as HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per. C6 cells, chicken embryo cells, or SF-9 cells can also be included, and these can be cultured in a variety of ways, including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as Wave bag systems. can. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293 cells adapted for growth in suspension culture. Numerous suspension cultures for producing rAAV particles are known in the art, including, for example, U.S. Pat. No. 20120122155, each of which is incorporated by reference in its entirety.

In some embodiments, the rAAV production culture comprises a high density cell culture. In some embodiments, the culture has a total cell density between about 1 x 10E+06 cells/ml and about 30 x 10E+06 cells/ml. In some embodiments, greater than about 50% of the cells are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293-derived cells (eg, HEK293T cells, HEK293F cells), Vero cells, or SF-9 cells. In further embodiments, the cells are HEK293 cells. In a further embodiment, the cells are HEK293 cells adapted for growth in suspension culture.

In additional embodiments of the provided methods, the rAAV production culture comprises a suspension culture containing rAAV particles. Numerous suspension cultures for producing rAAV particles are known in the art, including, for example, U.S. Pat. No. 20120122155, each of which is incorporated by reference in its entirety. In some embodiments, the suspension culture comprises a culture of mammalian or insect cells. In some embodiments, the suspension culture includes HeLa cells, HEK293 cells, HEK293-derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CHO cells, CHO-K1 cells, CHO-derived cells, EB66 cells, BSCs. cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per. Includes cultures of C6 cells, chicken embryo cells, or SF-9 cells. In some embodiments, the suspension culture comprises a culture of HEK293 cells.

In some embodiments, a method for the production of rAAV particles includes providing a cell culture comprising cells capable of producing rAAV, and adding to the cell culture between about 0.1 mM and about 20 mM. adding a histone deacetylase (HDAC) inhibitor to a final concentration and maintaining the cell culture under conditions that allow the production of the rAAV particles. In some embodiments, the HDAC inhibitor comprises short chain fatty acids or salts thereof. In some embodiments, the HDAC inhibitor comprises butyric acid (eg, sodium butyrate), valproic acid (eg, sodium valproate), propionic acid (eg, sodium propionate), or a combination thereof.

In some embodiments, rAAV particles are produced as disclosed in WO2020/033842, incorporated herein by reference in its entirety.

Recombinant AAV particles can be produced in a production culture containing host cells, provided that the cells are cultured under conditions known in the art to cause release of rAAV particles from intact host cells into the culture medium. rAAV can be harvested from rAAV production cultures by collecting spent media or by collecting spent media from production cultures. Recombinant AAV particles can also be harvested from rAAV production cultures by lysing the production culture's host cells. Suitable methods of lysing cells are also known in the art, such as multiple freeze/thaw cycles, sonication, microfluidization, and treatment with chemicals (e.g. detergents and/or proteases). can be mentioned.

At the time of harvest, the rAAV producing culture contains: (1) host cell proteins; (2) host cell DNA; (3) plasmid DNA; (4) helper virus; (5) helper virus proteins; (6) helper virus DNA. and (7) media components including, for example, serum proteins, amino acids, transferrin, and other low molecular weight proteins. The rAAV production culture can further contain product-associated impurities, such as inactive vector forms, empty viral capsids, aggregated viral particles or capsids, misfolded viral capsids, and degraded viral particles.

In some embodiments, the rAAV-producing culture collection is cleared to remove host cell debris. In some embodiments, the production culture collection is clarified by filtration through a series of depth filters. Clarification can also be performed by various other standard techniques known in the art, such as centrifugation, or any cellulose acetate with a pore size of 0.2 mm or more known in the art. This can be achieved by filtration with a filter. In some embodiments, clarification of the harvested cell culture includes sterile filtration. In some embodiments, the product culture collection is clarified by centrifugation. In some embodiments, clarification of the product culture collection does not include centrifugation.

In some embodiments, the harvested cell culture is clarified using filtration. In some embodiments, clarification of the harvested cell culture comprises depth filtration. In some embodiments, clarification of the harvested cell culture further includes depth filtration and sterile filtration. In some embodiments, the harvested cell culture is clarified using a filter train containing one or more different filtration media. In some embodiments, the filter train includes one depth filtration media. In some embodiments, the filter train includes one or more depth filtration media. In some embodiments, the filter train includes two depth filtration media. In some embodiments, the filter train includes one sterile filtration media. In some embodiments, the filter train includes two depth filtration media and one sterile filtration media. In some embodiments, the depth filter media is a porous depth filter. In some embodiments, the filter train includes Clarisolve® 20MS, Millistak+® COHC, and sterile grade filtration media. In some embodiments, the filter train includes Clarisolve® 20MS, Millistak+® COHC, and Sartopore® 2 XLG 0.2 μm. In some embodiments, the harvested cell culture is pretreated prior to contacting the depth filter. In some embodiments, pretreatment includes adding salt to the harvested cell culture. In some embodiments, pre-treatment includes adding a chemical flocculant to the harvested cell culture. In some embodiments, the harvested cell culture is not pretreated before contacting the depth filter.

In some embodiments, the product culture collection is clarified by filtration, as disclosed in WO 2019/212921, incorporated herein by reference in its entirety.

In some embodiments, the rAAV production culture collection is treated with a nuclease (e.g., Benzonase®) or an endonuclease (e.g., from Serratia marcescens) to digest the high molecular weight DNA present in the production culture. endonuclease). Nuclease or endonuclease digestion can be routinely performed under standard conditions known in the art. For example, nuclease digestion is performed at a final concentration of 1-2.5 units/mL of Benzonase® at temperatures ranging from ambient temperature to 37° C. for a period of 30 minutes to several hours.

Sterile filtration includes filtration with sterile grade filter media. In some embodiments, the sterile grade filtration media is a 0.2 or 0.22 μm pore filter. In some embodiments, the sterile grade filtration media comprises polyether sulfone (PES). In some embodiments, the sterile grade filtration media comprises polyvinylidene fluoride (PVDF). In some embodiments, the sterile grade filtration media has a hydrophilic heterogeneous bilayer design. In some embodiments, the sterile grade filtration media has a hydrophilic heterogeneous bilayer design with a 0.8 μm prefilter and a 0.2 μm final filter membrane. In some embodiments, the sterile grade filtration media has a hydrophilic heterogeneous bilayer design with a 1.2 μm prefilter and a 0.2 μm final filter membrane. In some embodiments, the sterile grade filtration media is a 0.2 or 0.22 μm pore filter. In a further embodiment, the sterile grade filtration media is a 0.2 μm pore filter. In some embodiments, the sterile grade filtration media is Sartopore® 2 It's a combination. In some embodiments, the sterile grade filtration media is Sartopore® 2 XLG 0.2 μm.

In some embodiments, the clarified feed is concentrated via tangential flow filtration (“TFF”) before being applied to a chromatography medium, such as an affinity chromatography medium. Large-scale concentrations of viruses using TFF ultrafiltration were determined by Paul et al. , Human Gene Therapy 4:609-615 (1993). The TFF concentration in the clarified feed allows technically manageable amounts of the clarified feed to be chromatographed and also allows for more rational column sizing without the need for long recirculation times. becomes possible. In some embodiments, the clarified feed is concentrated between at least 2 times and at least 10 times. In some embodiments, the clarified feed is concentrated between at least 10 times and at least 20 times. In some embodiments, the clarified feed is concentrated between at least 20 times and at least 50 times. In some embodiments, the clarified feed is concentrated about 20 times. Those skilled in the art will also appreciate that TFF is used to remove small molecule impurities (e.g., cell culture impurities including media components, serum albumin, or other serum proteins) from clarified feeds via diafiltration. You will also recognize that it is good. In some embodiments, the clarified feed is subjected to diafiltration to remove small molecule impurities. In some embodiments, diafiltration includes using between about 3 and about 10 diafiltration volumes of buffer. In some embodiments, diafiltration comprises using about 5 diafiltration volumes of buffer. Those skilled in the art will also recognize that TFF may be used in any step of the purification process where buffer exchange is desired before performing the next step in the purification process. In some embodiments, the methods for isolating rAAV from clarified feeds disclosed herein include the use of TFF to exchange buffer.

Affinity chromatography can be used to isolate rAAV particles from the composition. In some embodiments, affinity chromatography is used to isolate rAAV particles from the clarified feed. In some embodiments, affinity chromatography is used to isolate rAAV particles from a clarified feed that has been subjected to tangential flow filtration. Suitable affinity chromatography media are known in the art and include, but are not limited to, AVB Sepharose™, POROS™ CaptureSelect™ AAVX affinity resin, POROS™ CaptureSelect™ AAV9 affinity resin, and POROS™ CaptureSelect™ AAV8 affinity resin. In some embodiments, the affinity chromatography medium is POROS™ CaptureSelect™ AAV9 affinity resin. In some embodiments, the affinity chromatography medium is POROS™ CaptureSelect™ AAV8 affinity resin. In some embodiments, the affinity chromatography medium is POROS™ CaptureSelect™ AAVX affinity resin.

Anion exchange chromatography can be used to isolate rAAV particles from the composition. In some embodiments, anion exchange chromatography is used after affinity chromatography as a final concentration and polishing step. Suitable anion exchange chromatography media are known in the art and include, but are not limited to, UNOsphere™ Q (Biorad, Hercules, Calif.) and N-charged amino or imino resins, such as: POROS™ 50 PI, or any DEAE, TMAE, tertiary or quaternary amine, or PEI-based resin known in the art (U.S. Pat. No. 6,989,264; Brument et al. ., Mol. Therapy 6(5):678-686 (2002); Gao et al., Hum. Gene Therapy 11:2079-2091 (2000)). In some embodiments, the anion exchange chromatography medium includes a quaternary amine. In some embodiments, the anion exchange medium is a monolithic anion exchange chromatography resin. In some embodiments, the monolithic anion exchange chromatography media comprises a glycidyl methacrylate-ethylene dimethacrylate polymer or a styrene-divinylbenzene polymer. In some embodiments, the monolith anion exchange chromatography media is CIMmultus™ QA-1 Advanced Composite Column (Quaternary Amine), CIMmultus™ DEAE-1 Advanced Composite Column (Diethylamino), CIM® selected from the group consisting of QA Disk (quaternary amine), CIM® DEAE, and CIM® EDA Disk (ethylene diamino). In some embodiments, the monolith anion exchange chromatography media is a CIMmultus™ QA-1 Advanced Composite Column (Quaternary Amine). In some embodiments, the monolithic anion exchange chromatography media is a CIM® QA disk (quaternary amine). In some embodiments, the anion exchange chromatography medium is CIM QA (BIA Separations, Slovenia). In some embodiments, the anion exchange chromatography medium is BIA CIM® QA-80 (80 mL column volume). Those skilled in the art will appreciate that wash buffers of suitable ionic strength are used to ensure that impurities (including, but not limited to, impurities that may be introduced by upstream purification steps) are removed while the rAAV remains bound to the resin. It can be understood that it can be specified.

In some embodiments, anion exchange chromatography is performed according to the methods disclosed in WO2019/241535, incorporated herein by reference in its entirety.

In some embodiments, the method of isolating rAAV particles determines the vector genome titer, capsid titer, and/or complete capsid: empty capsid ratio in a composition comprising isolated rAAV particles. Including quantifying. In some embodiments, vector genome titer is quantified by quantitative PCR (qPCR) or digital PCR (dPCR) or droplet digital PCR (ddPCR). In some embodiments, capsid titer is quantified by serotype-specific ELISA. In some embodiments, the ratio of complete capsids to empty capsids is quantified by analytical ultracentrifugation (AUC) or transmission electron microscopy (TEM).

In some embodiments, vector genome titer, capsid titer, and/or complete capsid: empty capsid ratio is determined spectrophotometrically, e.g., by measuring the absorbance of the composition at 260 nm; and It is quantified by measuring the absorbance of the composition at 280 nm. In some embodiments, the rAAV particles are not denatured prior to measuring the absorbance of the composition. In some embodiments, the rAAV particles are denatured before measuring the absorbance of the composition. In some embodiments, the absorbance of the composition at 260 nm and 280 nm is determined using a spectrophotometer. In some embodiments, the absorbance of the composition at 260 nm and 280 nm is determined using HPLC. In some embodiments, the absorbance is peak absorbance. Several methods are known in the art for measuring the absorbance of compositions at 260 nm and 280 nm. Methods for quantifying vector genome and capsid titers of compositions comprising isolated recombinant rAAV particles are disclosed in WO2019/212922, incorporated herein by reference in its entirety.

In additional embodiments, the present disclosure provides compositions comprising isolated rAAV particles produced according to the methods disclosed herein. In some embodiments, the composition is a pharmaceutical composition that includes a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable" refers to a biologically acceptable formulation, gas, liquid, or Solids or mixtures thereof. A "pharmaceutically acceptable" composition is a material that is not biologically or otherwise undesirable, e.g., the material can be used without causing substantial undesirable biological effects. can be administered. Accordingly, such pharmaceutical compositions can be used, for example, in administering to a subject rAAV isolated according to the methods of the present disclosure. Such compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil) that are compatible with pharmaceutical administration or in vivo contact or delivery. ), suspensions, syrups, elixirs, dispersion and suspending media, coatings, isotonicity and absorption enhancers or retarders. Aqueous and non-aqueous solvents, solutions, and suspensions can contain suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules, and crystals. Supplementary active compounds such as preservatives, antimicrobial, antiviral, and antifungal agents can also be incorporated into the compositions. Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as described herein or as known to those skilled in the art. Accordingly, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes. Pharmaceutical compositions and delivery systems suitable for the rAAV particles and methods and uses of the invention are known in the art (see, eg, Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co. , Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms (1993), Technical Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calcul. (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al. , Drug Delivery Systems (1980), R.L. Juliano, ed., Oxford, N.Y., pp. 253-315).

In some embodiments, the composition is a pharmaceutical unit dose. "Unit dose" means physically discrete units suitable as unit dosages for the subject being treated. Each unit contains a predetermined amount, optionally together with a pharmaceutical carrier (excipient, diluent, vehicle, or filler), to produce the desired effect (e.g., prophylactic or therapeutic) when administered in one or more doses. effect). Unit dosage forms may be, for example, in ampoules and vials, which may contain liquid compositions or compositions in freeze-dried or lyophilized form, e.g., for administration in vivo using a sterile liquid carrier. or may be added prior to delivery. Individual unit dosage forms can be included in multi-dose kits or containers. Recombinant vector (e.g., AAV) sequences, plasmids, vector genomes, and recombinant viral particles, as well as pharmaceutical compositions thereof, can be formulated into single or multiple unit doses for ease of administration and uniformity of dosage. It can be packaged in any form. In some embodiments, the composition includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, and AAV. rAAV particles comprising AAV capsid proteins from AAV capsid serotypes selected from HSC16. In some embodiments, the AAV capsid serotype is AAV8. In some embodiments, the AAV capsid serotype is AAV9.

Example 1: AAV productivity of a process based on single-dose transient transfection in a 500L bioreactor was 48-60% of the productivity obtained using the 50L and 200L bioreactors.
The production of adeno-associated virus (AAV) from cell culture in the gene therapy field has evolved from simple adhesive flasks to complex adhesive systems to suspension-based stirred tank bioreactor processes. Many AAV gene therapy production systems exist, including transient transfection of mammalian cells (eg, suspension-adapted HEK293 cells). However, most of the equipment commonly used in modern floating mammal biotechnology processes is not specifically designed for large-scale transient production of AAV.

Comparing bioreactor scale and design magnifies the challenge of producing AAV in a suspension mammalian cell transfection process. Therefore, there is a need to compare current bioreactor systems for AAV production. The effects of bioreactor configuration and mixing on transfection and AAV production were compared. A 50L to 500L bioreactor was optimized for scalability and process performance. The effects of bioreactor settings and typical scale-up factors on cell growth, viability, and collected titers were compared in multiple types of bioreactors at various scales.

AAV-A vectors (containing AAV9 capsids) were generated via transient transfection of suspension-adapted HEK293 cells substantially as described herein. Briefly, suspension-adapted HEK293 cells were thawed and expanded. Bioreactors were seeded with suspension-adapted HEK293 cells at a density of approximately 1.0-1.2×10 ⁶ viable cells/mL. At 72 hours ECD (lapse culture period), cells were transfected with a mixture of polyethyleneimine (PEI) and three plasmids encoding adenoviral helper functions, an AAV-A transgene, and AAV9 Cap/Rep. Transfections were performed using a DNA:PEI ratio of 1:1.75. Transfection complex preparation process flow diagrams for 20L, 200L, and 500L bioreactors are shown in Figures 1-3. DNA and PEI were mixed using an in-line mixer. The volume of transfection complex added was approximately 10% of the volume of the cell culture. Culture supernatants were collected at 144-192 hours ECD (eg, 3-5 days after transfection). The AAV-A yield obtained is shown in FIG. Relative titers were determined as is customary in the art.

First generation processes up to 500L scale in multi-vendor bioreactors (vendor A or B) show linear cell growth parameters up to production initiation (e.g., up to the transient transfection step). (data not shown). This indicated that P/V and Tip Speed were the optimal scaling parameters for cell growth. During the production phase, cell growth and viability followed similar trends in the 50L and 200L bioreactors. The collection VCD and survival rate of 500L was actually higher than 50L and 200L. This indicates low transfection efficiency. Considering that cell growth and viability were similar in multiple bioreactors tested by two different vendors, the productivity of the 500L bioreactor for AAV-A was the average of the last four production runs. It was surprising that it was only 48-60% based on In contrast, the productivity of the 50L and 200L bioreactors was in line with the average. Figure 4.

Example 2: Complexation time affects AAV productivity It has been reported that DNA and PEI complexation time affects the size of the complexes formed; may affect transfection efficiency. As shown in Figure 5, increasing the complexation time resulted in the formation of larger DNA:PEI complexes. Complex size was measured using dynamic light scattering using known methods.

The experimental schematic in Figure 6 was used to test the effect of increasing conjugation time on AAV-A productivity. Briefly, a 42L transfection complex of PEI and a (3) plasmid encoding adenovirus helper functions, the AAV-A transgene, and AAV9 Cap/Rep was mixed together with the DNA and PEI via an in-line mixer. It was prepared by Once mixed, the transfection complexes were allowed to incubate. Fixed volumes of transfection complexes were then removed at predetermined time points (10, 20, 40, 60 minutes after the start of complex incubation) and used to transfect bioreactors and shake flasks.

As shown in Figure 7, increasing complexation time decreased AAV productivity. The results of this experiment show that GC titers 4 and 5 days after transfection steadily decreased with increasing transfection complex time.

Example 3: Single-dose and split-transient transfection-based processes were comparable in AAV productivity.
Without wishing to be bound by any particular theory, one possible explanation for the low productivity of AAV-A observed in Example 1 using the 500 L bioreactor is that mixing, conjugation, and This is likely due to constraints on incubation and adding the required volume of transfection complex (42 L for a 500 L bioreactor). As shown in Example 2, the size of DNA:PEI complexes increases with time and using DNA:PEI complexes above the optimal size reduces AAV-A productivity. The combination of a narrow operating window of approximately 30 minutes and a large volume of transfection complex (42 L) resulted in low yields due to the fact that incubation and pumping time for this large volume of transfection complex was potentially insufficient. (50-60% of the prediction).

We tested a process based on split-transient transfection in which smaller volumes of transfection complexes were mixed, complexed, and added to the cell culture instead of a single large volume of the transfection process. We determined whether the partitioning of 2.0 into 2 or more steps could be used to overcome constraints regarding transfection complex volume and narrow operating windows. A 200L bioreactor process was used to quantify the productivity of AAV-B (including AAV8 capsids) for single-dose and split-transient transfection-based processes. The single-dose transient transfection-based process was carried out substantially as described in Example 1. A transfection complex preparation process flow diagram in a split transfection-based process is shown in FIG. The total volume of transfection complex added was virtually the same (approximately 16 L) for single-dose and split transfection processes. Whereas the single-dose process involves mixing, complexing, and adding the total volume in one step, the split transfection process involves two separate mixing, Complexing and addition steps were used, with the second addition step starting 15-30 minutes after completing the first addition step. Culture supernatants were collected at 168 hours ECD, 4 days after transfection. The AAV-B yield obtained is shown in FIG. The split transient transfection process yielded similar titers as runs using a single dose of the transfection complex. This was an important finding because it addresses the narrow operating window (30 minutes) in the preparation and addition of transfection complex volumes for large (eg, 200 L or larger) bioreactors. Dividing the transfection complex into smaller doses allows for the use of more robust complex incubation times and pump flow rates within a 30 minute operating window for each dose.

Example 4: Split transfection complex doses can be added at intervals of several hours without significantly compromising AAV productivity.
A 2L bench scale experiment with AAV-B was performed to test the robustness of the split transient transfection process. The objective was to see how far apart the addition of split transfection complexes could be spaced and still obtain comparable GC titers. The interval between split additions of transfection complexes was varied from 15 minutes to 6 hours. Cell growth and viability trends were similar for the conditions tested (data not shown). Glucose trends were also similar between conditions (data not shown). The trends for L-glutamine and NH4+ were similar up to 144 hours ECD for all conditions tested. From this point on, bioreactors that allowed 15 minutes between split additions of transfection complexes consumed more L-glutamine and produced more NH4+. This difference did not adversely affect production. This is because these bioreactors showed comparable or in some cases higher GC titers than bioreactors with 4 and 6 hours between separate additions of transfection complexes. Figure 10. Although slightly greater GC titer variability was observed, split addition of transfection complexes to the bioreactor up to 6 h apart can still yield titers within 80-90% of the predicted range. can.

Example 5: AAV-B productivity of a process based on split transient transfection in a 500L bioreactor is similar to that obtained using 50L and 200L bioreactors.
A planktonic HEK293 clone was used to test the AAV-B productivity of a process based on split transient transfection in a 500 L bioreactor. A flow diagram of the transfection complex preparation process in a split transfection-based process is shown in FIG. Two doses of approximately 21 L of transfection complex were added to the culture with 15-30 minutes between additions. Culture supernatants were collected at 168 hours ECD, 4 days after transfection. The relative titer of AAV-B produced is shown in FIG. The productivity of the 500L bioreactor was similar to both the "historical average" and the productivity obtained using the single dose transfection process in the 200L and 50L bioreactors.

Example 6: AAV-B productivity of a process based on split transient transfection in a 200L scale-down of a 2,000L process.
AAV-B productivity in a 200L scale-down of a 2,000L split transient transfection-based process was tested in a 200L bioreactor. An anti-aggregating agent was included in the seed train medium. Four doses of transfection complex of approximately 4 L were added to the culture with approximately 15 minutes between additions. A process flow diagram is shown in FIG. Culture supernatants were collected at 168 hours ECD, 4 days after transfection. The titer of the supernatant was approximately 6.25E+10. The solubility titer was approximately 1.15E+11.

Example 7: AAV productivity of a process based on 2,000L split transient transfection.
In some embodiments, a process based on 2,000 L split transient transfection involves dividing 4 x about 42 L transfection complexes into 2,000 L cells containing about 1600 L of cell culture (e.g., HEK cell culture). including transfer to a bioreactor. In some embodiments, the culture includes an anti-aggregating agent. In some embodiments, the transfection complex is produced by mixing the diluted one or more polynucleotides and the diluted at least one transfection reagent using an in-line mixer; , transferring the diluted one or more polynucleotides and the diluted at least one transfection reagent from two separate containers to a new container at a rate of about 8 liters/minute. In some embodiments, the transfection complex is held for 10-20 minutes (eg, 10-15 minutes) before transferring to the bioreactor. In some embodiments, the transfection complex is transferred to the bioreactor at a rate of about 5 L/min. In some embodiments, each batch of transfection complexes is transferred to a bioreactor in 30 minutes or less. In some embodiments, it takes 10-20 minutes (e.g., about 15 minutes) between finishing transferring one batch of transfection complexes and starting transferring the next batch of transfection complexes. ) is considered to be a gap.

Although the methods of the present disclosure have been described in conjunction with what are presently considered to be the most practical and preferred embodiments, the methods encompassed by the present disclosure should not be limited to the disclosed embodiments; on the contrary, , is intended to cover various modifications and equivalent arrangements that may come within the spirit and scope of the appended claims.

All publications, patents, patent applications, Internet sites, and accession numbers/database sequences (including both polynucleotide and polypeptide sequences) cited herein are referred to in each individual publication. The patent, patent application, Internet site, or accession number/database sequence is herein incorporated by reference in its entirety for all purposes to the same extent as if the patent, patent application, Internet site, or accession number/database sequence were specifically and individually incorporated by reference. Ru.

Claims (58)

1. A method of producing recombinant virus particles, the method comprising:
a) providing a suspension cell culture of between about 200 liters and about 20,000 liters containing a population of cells capable of producing said recombinant virus particles;
b) combining one or more polynucleotides with at least one transfection reagent to form a first mixture, incubating said mixture to form a polynucleotide:transfection reagent complex; transferring the polynucleotide:transfection reagent complex to the suspension culture to transfect cells;
c) combining said one or more polynucleotides with said at least one transfection reagent to form a second mixture and incubating said mixture to form a polynucleotide:transfection reagent complex; , transferring the polynucleotide:transfection reagent complex to the suspension culture to transfect the cells;
d) maintaining said cell culture containing said transfected cells under conditions that allow production of said recombinant virus particles;
(i) said one or more polynucleotides include genes necessary for the production of said recombinant viral particles;
(ii) the mixing, the incubating, and the transferring of steps b) and c) are each completed in less than about 60 minutes;
(iii) said transferring of step b) and step c) is carried out over a period of about 6 hours or less, and (iv) said transferring of step b) and step c) is performed simultaneously or sequentially in any order. carried out,
Said method. 2. The method of claim 1, wherein step c) is repeated one more time. 2. The method of claim 1, wherein step c) is repeated one or more times. 2. The method of claim 1, wherein step c) is repeated 1, 2, 3, 4, 5, 6, 7, or 8 times. 2. The combined volume of the polynucleotide:transfection reagent complex transferred to the suspension culture is between about 5% and about 20% of the volume of the suspension cell culture of step a). 4. The method according to any one of items 4 to 4. 6. A method according to any preceding claim, wherein said transferring of step c) is initiated between about 5 minutes and about 60 minutes after completing said transferring of step b). 6. A method according to any preceding claim, wherein said transferring of step c) is initiated within about 60 minutes of completing said transferring of step b). A method of increasing the production of recombinant virus particles, the method comprising:
a) providing a suspension cell culture of between about 200 liters and about 20,000 liters containing a population of cells capable of producing said recombinant virus particles;
b) combining one or more polynucleotides with at least one transfection reagent to form a first mixture; incubating said mixture to form a polynucleotide:transfection reagent complex; transferring the polynucleotide:transfection reagent complex to the suspension culture to transfect cells;
c) combining said one or more polynucleotides with said at least one transfection reagent to form a second mixture and incubating said mixture to form a polynucleotide:transfection reagent complex; , transferring the polynucleotide:transfection reagent complex to the suspension culture to transfect the cells;
d) maintaining said cell culture containing said transfected cells under conditions that allow production of said recombinant virus particles;
(i) said one or more polynucleotides include genes necessary for the production of said recombinant viral particles;
(ii) the mixing, the incubating, and the transferring of steps b) and c) are each completed in less than about 60 minutes;
(iii) said transferring of step b) and step c) is performed over a period of about 6 hours or less, and (iv) said transferring of step b) and step c) is performed simultaneously or consecutively in any order. carried out,
Said method. 9. The method of claim 8, wherein step c) is repeated one more time. 9. The method of claim 8, wherein step c) is repeated one or more times. 9. The method of claim 8, wherein step c) is repeated 1, 2, 3, 4, 5, 6, 7, or 8 times. 9. The combined volume of the polynucleotide:transfection reagent complex transferred to the suspension culture is between about 5% and about 20% of the volume of the suspension cell culture of step a). The method according to any one of items 1 to 11. 13. A method according to any one of claims 8 to 12, wherein the transferring of step c) is initiated between about 5 minutes and about 60 minutes after completing the transferring of step b). 14. A method according to any one of claims 8 to 13, wherein the transferring of step c) is started within about 60 minutes of completing the transferring of step b). 6. The method of any one of the preceding claims, wherein mixing the one or more polynucleotides with at least one transfection reagent is performed by an in-line mixer. 11. The method of any preceding claim, wherein the mixing, incubating, and transferring of steps b) and c) are each completed in less than about 30 minutes. 5. The method of any preceding claim, wherein the mixing, incubating, and transferring of steps b) and c) are each completed in less than about 35 minutes. The method of any one of the preceding claims, wherein the incubating steps of step b) and step c) are each for about 10 to about 20 minutes. The method of any one of the preceding claims, wherein the incubating steps of step b) and step c) are each for about 10 to about 15 minutes. 7. The method of any one of the preceding claims, wherein the at least one transfection reagent comprises a stable cationic polymer. 7. The method of any one of the preceding claims, wherein the at least one transfection reagent comprises PEI. 5. The method of any preceding claim, wherein the cell culture has a volume of between about 200 liters and about 5,000 liters. 5. The method of any preceding claim, wherein the cell culture has a volume of between about 200 liters and about 2,000 liters. 7. The method of any preceding claim, wherein the cell culture has a volume of between about 200 liters and about 1,000 liters. 6. The method of any preceding claim, wherein the cell culture has a volume of between about 200 liters and about 500 liters. The cell culture is about 200 liters, about 300 liters, about 400 liters, about 500 liters, about 750 liters, about 1,000 liters, about 2,000 liters, about 3,000 liters, or about 5,000 liters. A method according to any one of the preceding claims, having a volume of . 5. A method according to any preceding claim, wherein the cell culture has a volume of approximately 500 liters. 10. The method of any one of the preceding claims, wherein the cell culture has a volume of approximately 1,000 liters. 5. The method of any preceding claim, wherein the cell culture has a volume of approximately 2,000 liters. 11. The method of any one of the preceding claims, wherein the population of cells comprises a population of mammalian cells or insect cells. 5. The method of any one of the preceding claims, wherein the population of cells comprises a population of mammalian cells. Any of the preceding claims, wherein the population of cells comprises a population of HEK293 cells, HEK-derived cells, CHO cells, CHO-derived cells, HeLa cells, SF-9 cells, BHK cells, Vero cells, and/or PerC6 cells. The method described in Section 1. 5. The method of any one of the preceding claims, wherein the population of cells comprises a population of HEK293 cells. 5. The method of any one of the preceding claims, wherein the suspension cell culture provided in step a) contains between about 2x10E+6 and about 10E+7 viable cells/ml. under conditions that allow said cell culture to produce said recombinant virus particles for about 2 days to about 10 days, about 3 days to about 5 days, or about 5 days to 14 days. A method according to any one of the preceding claims, wherein the method is maintained. 11. The method of any one of the preceding claims, wherein the cell culture is maintained for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. 3. The method of any one of the preceding claims, wherein the cell culture is maintained for at least about 3 days. 3. The method of any one of the preceding claims, wherein the cell culture is maintained for about 3 days. 5. The method of any one of the preceding claims, wherein the cell culture is maintained for about 4 days. 5. The method of any one of the preceding claims, wherein the recombinant virus particles are recombinant adeno-associated virus (rAAV) particles or recombinant lentiviral particles. 5. The method of any one of the preceding claims, wherein the recombinant virus particles are rAAV particles. The rAAV particles may be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, or AAV. 42. The method of claim 41, comprising a capsid protein of serotype HSC16. The rAAV particles may be AAV8, AAV9, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, or AAV. 42. The method of claim 41, comprising capsid protein of hu37 serotype. 42. The method of claim 41, wherein the rAAV particle comprises a capsid protein of AAV8 or AAV9 serotype. 45. The method of any one of claims 41-44, wherein the rAAV particle comprises a genome containing a transgene. 46. The method of claim 45, wherein the transgene comprises a regulatory element operably connected to a polynucleotide encoding a polypeptide. 47. The method of claim 46, wherein the regulatory elements include one or more of an enhancer, a promoter, and a polyA region. 47. The method of claim 45 or claim 46, wherein the regulatory element and the polynucleotide encoding the polypeptide are heterologous. 49. According to any one of claims 45 to 48, the transgene encodes an antibody or antigen binding fragment thereof, a fusion protein, an Fc fusion polypeptide, an immunoadhesin, an immunoglobulin, an engineered protein, a protein fragment, or an enzyme. Method described. The transgene may be an anti-VEGF Fab, iduronidase (IDUA), iduronate 2-sulfatase (IDS), low density lipoprotein receptor (LDLR), tripeptidyl peptidase 1 (TPP1), or VEGF receptor 1 (sFlt-1). 49. The method according to any one of claims 45 to 48, encoding a non-membrane bound splice variant of ). The transgene is gamma-sarcoglycan, Rab escort protein 1 (REP1/CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide gated channel alpha 3 (CNGA3), cyclic nucleotide gated channel beta 3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), factor VIII, factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinosxin (RS1), sarcoplasmic reticulum calcium ATP ase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamate decarboxylase (GAD), glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, microdystrophin , myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), arylsulfatase B (ARSB) ), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha 1-antitrypsin (AAT), Phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neurturin (NRTN), neurotrophin-3 (NT-3/NTF3), porphobilinogen deaminase ( PBGD), nerve growth factor (NGF), mitochondrial code NADH: ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dysferlin, MER proto-oncogene tyrosine kinase (MERTK), cystic 49. The method of any one of claims 45-48, encoding a fibrosis transmembrane conductance regulator (CFTR) or a tumor necrosis factor receptor (TNFR)-immunoglobulin (IgG1) Fc fusion. said one or more polynucleotides,
a) rAAV genome to be packaged;
b) adenovirus helper functions necessary for packaging;
52. The method of any one of claims 41-51, encoding: c) sufficient AAV rep protein for packaging; and d) sufficient AAV cap protein for packaging. 3. The one or more polynucleotides comprising a polynucleotide encoding the rAAV genome, a polynucleotide encoding the AAV rep protein and the AAV cap protein, and a polynucleotide encoding the adenovirus helper function. The method according to item 52. 54. The method of claim 52 or 53, wherein the adenovirus helper function comprises at least one of an adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene. 55. The method of any one of claims 41-54, further comprising collecting the rAAV particles. 56. The method of any one of claims 41-55, wherein the cell culture produces between about 5 x 10e+10 GC/ml and about 1 x 10e+12 GC/ml of rAAV particles. the cell culture produces at least about twice as many rAAV particles, measured as GC/ml, than a reference method involving a single step of mixing, incubating, and transferring the same volume of polynucleotide:transfection reagent complexes; 57. The method according to any one of claims 41 to 56. A composition comprising isolated rAAV particles produced by the method of any one of claims 41-57.

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